Anti-cdh3 antibodies and uses thereof

ABSTRACT

The present invention relates to anti-CDH3 antibodies, which can be labeled with a radioisotope. Moreover, the present invention provides methods and pharmaceutical compositions that comprise an anti-CDH3 antibody as an active ingredient. Since CDH3 is strongly expressed in pancreatic, lung, colon, prostate, breast, gastric or liver cancer cells, the present invention is useful in pancreatic, lung, colon, prostate, breast, gastric or liver cancer therapies.

TECHNICAL FIELD

The present invention relates to anti-CDH3 antibodies, methods fortreating or preventing, or diagnosing a disease associated with CDH3using the antibodies, and pharmaceutical compositions or diagnosing kitscomprising the antibodies.

BACKGROUND ART

Cadherins are cell-cell adhesion glycoproteins that formcalcium-dependent inter-cellular junctions and play an essential role inmorphogenesis and in the development and maintenance of adult tissuesand organs (NPL 1). During embryogenesis, the cell expression ofspecific cadherins results in homophilic interactions that are criticalin the process of cell sorting and tissue stratification (NPL 2-4).Alterations in these cellular attachments play an important role in celldestabilization and may modify the carefully regulated differentiationprocess of the epithelial structures (NPL5-6). For this reason, thefunctional loss or overexpression of cadherins and the molecularmechanisms underlying the control of the genes codifying these proteinshave been implicated in carcinogenesis (NPL 7).

The cadherin family is subdivided into various subfamilies, includingthe classical E-, P-, and N-cadherins, each demonstrating a specifictissue distribution (NPL 8). Although E-cadherin is expressed in allepithelial tissues, the expression of P-cadherin (CDH3) is onlyrestricted to the basal or lower layers of stratified epithelia,including prostate and skin, and also to the breast myoepithelial cells(NPL 9-10).

A large body of evidence now also reveals that aberrant P-cadherinexpression is associated with cell proliferation and with tumors of thecolon, breast, lung, thyroid, and cervix (NPL 11-12). Human P-cadherinwas reported to be the antigen recognized by the NCC-CAD-299 monoclonalantibody raised against a vulvar epidermoid carcinoma (NPL 10).Modulation of P-cadherin mediated adhesion and intracellular signalingis expected to result in decreased proliferation and survival of tumorcells in vivo. Accordingly, in view of the pivotal role that P-cadherinappears to possess in cell proliferation and solid tumor progression, itis desirable to generate antibodies to P-cadherin that can provide atherapeutic benefit to patients with a variety of cancers.

Monoclonal antibodies against cancer-specific molecules have been provedto be useful in cancer treatment (NPL 13). In addition to successfulexamples of clinical application of the humanized or chimeric antibodiessuch as trastuzumab (NPL 14), rituximab (NPL 15) and bevacizumab (NPL16) for breast cancer, malignant lymphoma and colon cancer, a number ofmonoclonal antibodies against other molecular targets are in developmentand being evaluated their anti-tumor activities. These monoclonalantibodies are expected to provide a hope to patients having tumors thathave no effective treatment. One of the other important issues for thesemonoclonal antibodies is achievement of selective therapeutic effects tocancer cells without severe toxicity due to their specific reaction tocells expressing target molecules (NPL 17-19, PTL 1-4).

CITATION LIST Patent Literature

-   PTL 1: WO2002/097395-   PTL 2: WO2004/110345-   PTL 3: WO2006/114704-   PTL 4: WO2007/102525

Non Patent Literature

-   NPL 1: Conacci-Sorrell M, et al., J Clin Invest, 109:987-91, (2002-   NPL 2: Nose A, et al., Cell, 54:993-1001, (1988)-   NPL 3: Steinberg M S, et al., Proc Natl Acad Sci USA, 91:206-9,    (1994)-   NPL 4: Takeichi M. Science, 251:1451-5, (1991)-   NPL 5: Daniel C W, et al., Dev Biol, 169:511-9, (1995)-   NPL 6: Nose A and Takeichi M. J Cell Biol, 103:2649-58, (1986)-   NPL 7: Behrens J. Cancer Metastasis Rev, 18:15-30 (1999)-   NPL 8: Takeichi M. Development, 102:639-55 (1988)-   NPL 9: Takeichi M. J Cell Biol 103:2649-58, (1986)-   NPL 10: Shimoyama Y, et al., Cancer Res, 49:2128-33 (1989)-   NPL 11: Gamallo, Modern Pathology, 14:650-654, (2001)-   NPL 12: Stefansson, et al., J. Clin. Oncol. 22(7):1242-1252 (2004)-   NPL 13: Harris, M. Lancet Oncol, 5: 292-302 (2004)-   NPL 14: Baselga, J. Oncology, 61: SuuplSuupl 2 14-21 (2004)-   NPL 15: Maloney, D. G., et al. Blood, 90: 2188-2195 (1997)-   NPL 16: Ferrara, N., et al. Nat Rev Drug Discov, 3: 391-400 (2004)-   NPL 17: Crist, W. M., et al. J Clin Oncol, 19: 3091-3102 (2001)-   NPL 18: Wunder, J. S., et al. J Bone Joint Surg Am, 80: 1020-1033    (1998)-   NPL 19: Ferguson, W. S. and Goorin, A. M. Cancer Invest, 19: 292-315    (2001)

SUMMARY OF INVENTION

The present invention provides monoclonal antibodies against CDH3, whichspecifically recognize a CDH3 polypeptide such as a polypeptide havingthe amino acid sequence as shown in SEQ ID NO: 2 or a fragment thereof.The present invention provides an evidence that 90Y-labeled anti-CDH3monoclonal antibodies have significant antitumor effect in xenograftmice bearing a cancer cell line.

Specifically, the present invention relates to the following:

[1] An antibody or a fragment thereof, wherein the antibody comprises anH (heavy) chain V (variable) region and an L (light) chain V region,wherein the H chain V region and the L chain V region selected from thegroup consisting of:

(a) an H chain V region comprising complementarity determining regions(CDRs) included in an H chain V region having the amino acid sequenceshown in SEQ ID NO: 4 or CDRs functionally equivalent thereto, and an Lchain V region comprising CDRs included in an L chain V region havingthe amino acid sequence shown in SEQ ID NO 12 or CDRs functionallyequivalent thereto;

(b) an H chain V region comprising CDRs included in an H chain V regionhaving the amino acid sequence shown in SEQ ID NO: 20 or CDRsfunctionally equivalent thereto, and an L chain V region comprising CDRsincluded in an L chain V region having the amino acid sequence shown inSEQ ID NO: 28 or CDRs functionally equivalent thereto;

(c) an H chain V region comprising CDRs included in an H chain V regionhaving the amino acid sequence shown in SEQ ID NO: 36 or CDRsfunctionally equivalent thereto, and an L chain V region comprising CDRsincluded in an L chain V region having the amino acid sequence shown inSEQ ID NO 44 or CDRs functionally equivalent thereto; and

(d) an H chain V region comprising CDRs included in an H chain V regionhaving the amino acid sequence shown in SEQ ID NOs: 68, 72, 76 or 80, orCDRs functionally equivalent thereto, and an L chain V region comprisingCDRs included in an L chain V region having the amino acid sequenceshown in SEQ ID NO: 60 or CDRs functionally equivalent thereto,

and wherein the antibody is capable of binding to a CDH3 polypeptide ora partial peptide thereof.

In typical embodiments, the antibody is selected from the groupconsisting of a mouse antibody, a chimeric antibody, a humanizedantibody, a human antibody, an antibody fragment, and single-chainantibody.

[2] The antibody or fragment thereof of the present invention can beconjugated with a cytotoxic, a therapeutic agent, a radioisotope labelor a fluorescent label. In typical embodiments, the antibody is labeledwith a radioisotope label. In more typical embodiments, the radioisotopelabel is selected from the group consisting of 90yttrium (⁹⁰Y),125iodine (¹²⁵I) and 111indium (¹¹¹In).

[3] A method for treating or preventing a disease associated with CDH3,or inhibiting CDH3-expressing cells grow, in a subject, the methodcomprising administering to the subject an effective amount of theantibody or fragment thereof of the present invention.

[4] A method for diagnosis or prognosis of a disease that is associatedwith CDH3 or of a predisposition to develop the disease in a subject,comprising

(a) contacting a sample or a specimen from the subject with the antibodyor fragment of the invention;(b) detecting a CDH3 polypeptide in the sample or specimen; and(c) determining whether or not the subject suffers from or is at risk ofdeveloping the disease based on the relative abundance of the CDH3polypeptide compared to a control.

[5] A pharmaceutical composition, for treating or preventing a diseaseassociated with CDH3, or inhibiting CDH3-expressing cells growth, thecomposition comprising an effective amount of the antibody or fragmentthereof of the present invention, and a pharmaceutically acceptablecarrier or excipient.

[6] A kit for diagnosis or prognosis of a disease associated with CDH3,the kit comprising the antibody or fragment of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the effect of 90Y-labeled anti-CDH3 antibodies (clone #3,#4, #5 and #6) on tumor growth. 90Y-labeled anti-CDH3 antibodies, inparticular clone #3, #4 and #6, effectively suppressed the growth ofH1373 cells grafted in nude mice, while no suppression of tumor growthwas demonstrated by administration of non-labeled anti-CDH3 antibodies.

FIG. 2 shows reduced SDS-PAGE analysis of anti-CDH3 antibodies (clone#3, #4, #5 and #6). Banding pattern of clone #3, #4 and #5 revealed twobands corresponding to IgG heavy chain and light chain, while that ofclone#6 revealed two heavy chain bands and single light chain band.

DESCRIPTION OF EMBODIMENTS

The present invention relates to anti-CDH3 antibodies, compositionscomprising them and their use in treating or preventing a diseaseassociated with CDH3 such as cancer. In a typical embodiment, theantibody is labeled with a radioisotope label.

cDNA microarrays for gene expression analysis of pancreatic cancer cellsand normal cells collected from pancreatic cancer patients has beenreported (Nakamura et al., (2004) Oncogene; 23: 2385-400). A number ofgenes with specifically enhanced expression in pancreatic cancer cellswere subsequently identified. Placental cadherin (P-cadherin; CDH3),which is a cytoplasmic membrane protein, was one of these genes andrepresented low levels of expression in major organs. Such features aresuitable for a target gene for cancer therapy because the danger of sideeffects will be avoided. In addition, a similar over-expression of CDH3was confirmed in other cancer cell lines, such as the lung, colorectal,prostate, breast, gastric and liver-cancer cell lines (WO/2007/102525).

DEFINITION

The terms “antibody” as used herein is intended to includeimmunoglobulins and fragments thereof which are specifically reactive tothe designated protein or peptide thereof. An antibody can include humanantibodies, primatized antibodies, chimeric antibodies, bispecificantibodies, humanized antibodies, antibodies fused to other proteins orradiolabels, and antibody fragments. Furthermore, an antibody herein isused in the broadest sense and specifically covers intact monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies) formed from at least two intact antibodies, andantibody fragments so long as they exhibit the desired biologicalactivity. An “antibody” indicates all classes (e.g. IgA, IgD, IgE, IgGand IgM).

“Antibody fragments” is a portion of an intact antibody, generallycomprises one or more antigen binding or variable regions of the intactantibody. Accordingly, in the present invention, antibody fragments maycomprise one or more antigen binding portions of the intact antibody.The term “antigen-binding portion” of an antibody, as used herein,refers to one or more immunological active fragments of an antibody thatretain the ability to specifically bind to an antigen (e.g., CDH3). Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Examples of antibodyfragments include Fab, Fab′, F(ab′)2, and Fv fragments; linearantibodies; and single chain antibody molecules. Regardless ofstructure, an antibody fragment binds with the same antigen that isrecognized by the intact antibody. The term “antibody fragment” alsoincludes a synthetic or a genetically engineered polypeptide that bindsto a specific antigen, such as polypeptides consisting of the lightchain variable region, “Fv” fragments consisting of the variable regionsof the heavy and light chains, recombinant single chain polypeptidemolecules in which light and heavy variable regions are connected by apeptide linker (“scFv proteins”), and minimal recognition unitsconsisting of the amino acid residues that mimic the hypervariableregion.

The term “functionally equivalent” to the CDRs as used herein, refers toCDRs that give an antibody having them substantially equalantigen-binding activity and antigen specificity to the antibody havingoriginal CDRs. Examples of functionally equivalent to the CDRs includethose wherein one or more amino acid residues are substituted, deleted,inserted and/or added to the original CDRs. So long as the antigenbinding activity and antigen specificity are maintained, the number orpercentage of amino acid mutations are not particularly limited.However, it is generally preferred to alter 20% or less of the aminoacid sequence of original CDRs included in a variable region. Morepreferably, percentage of mutations is 15% or less, 10% or less, 5% orless. In preferred embodiment, the number of amino acid mutations is 10or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 or less. Oneskilled in the art will understand acceptable mutations which retain theproperties of the original CDRs.

Production of Antibodies

The present invention uses monoclonal anti-CDH3 antibodies. Theseantibodies will be provided by methods well known in the art.

Exemplary techniques for the production of the antibodies used inaccordance with the present invention are described below.

(i) Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature, 256: 495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized with a CDH3 polypeptide to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to CDH3 polypeptides. Alternatively, lymphocytes maybe immunized with a CDH3 polypeptide in vitro. Lymphocytes then arefused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells prepared are seeded and grown in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. For example,if the parental myeloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas can typically include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Preferredmyeloma cell lines include murine myeloma lines, such as those derivedfrom MOPC-21 and MPC-11 mouse tumors available from the Salk InstituteCell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653cells available from the American Type Culture Collection, Manassas,Va., USA. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodies(Kozbor, J. Immunol., 133: 300 1 (1984); Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp. 51-63 (MarcelDekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the 30 Scatchard analysis of Munson et al., Anal.Biochem., 107: 220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPML-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5: 256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151-188 (1992).

Another method of generating specific antibodies, or antibody fragments,reactive against a CDH3 is to screen expression libraries encodingimmunoglobulin genes, or portions thereof, expressed in bacteria with aCDH3 protein or peptide. For example, complete Fab fragments, VH regionsand Fv regions can be expressed in bacteria using phage expressionlibraries. See for example, Ward et al., Nature 341: 544-546 (1989);Huse et al., Science 246: 1275-1281 (1989); and McCafferty et al.,Nature 348: 552-554 (1990). Screening such libraries with, for example,a CDH3 peptide, can identify immunoglobulin fragments reactive withCDH3. Alternatively, the SCID-hu mouse (available from Genpharm) can beused to produce antibodies or fragments thereof.

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348: 552-554 (1990). Clackson etal., Nature, 352: 624-628 (1991) and Marks et al., J MoL Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies,respectively, using phage libraries. Subsequent publications describethe production of high affinity (nM range) human antibodies by chainshuffling (Marks et al., BioTechnology, 10: 779-783 (1992)), as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., Nuc. Acids.Res., 21: 2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl. Acad. ScL USA, 81: 6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically, such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

The present invention provides antibodies suitable for treating and/orpreventing a disease, and/or inhibiting CDH3-expressing cells growth.The present invention also provides antibodies suitable for diagnosingCDH3 associated disease. In the present invention, murine monoclonalantibodies clone #3, #4 and #5 are successfully established and theseantibodies, in particular clone #3, were demonstrated to effectivelysuppress CDH3-expressing cells (e.g., cancer cells) growth. Further,these clones have no glycosylation sites within their variable regions.This property is an advantage for developing of therapeutic drugs sinceit may support uniformity of the antibody.

Also, in the present invention, variants, which have no glycosylationsites, of clone #6 are successfully established. Clone #6 have beendemonstrated as a antibodies which has capability of inhibit variousCDH3-expressing cells growth, however, it has a glycosylation sitewithin CDR2 of the H chain V region. The variants of the presentinvention are changed an asparagine residue of glycosylation site to aserine, threonine, alanine or glutamine residue.

The amino acid sequences of H chain V region of mouse monoclonalantibodies clone #3, clone #4 and #5 are shown in SEQ ID NOs: 4, 20, 36,respectively. The amino acid sequences of L chain V region of mousemonoclonal antibodies clone #3, clone #4 and #5 are shown in SEQ ID NOs:12, 28, 44, respectively. Further, the amino acid sequences of H chain Vregion of variants of clone #6 are shown in SEQ ID NOs: 68, 72, 76 and80, respectively. The amino acid sequences of L chain V region ofvariants of clone #6 are shown in SEQ ID NO: 60.

CDRs included in an H chain V region and an L chain V region can bedetermined according to methods well-known in the art. For example, themethods described by Kabat et al (Kabat E. A. et al. (1991) Sequence ofProteins of Immunological Interest. 5th Edition) or Chothia et al(Chothia et al. J. Mol. Biol. (1987) 196; 901-917) are generally usedfor CDRs determination.

Therefore, the present invention provides antibodies or fragmentsthereof, comprising an H chain V region and an L chain V region, whereinthe H chain V region and the L chain V region selected from the groupconsisting of:

(a) an H chain V region comprising CDRs included in an H chain V regionhaving the amino acid sequence shown in SEQ ID NO: 4 or CDRsfunctionally equivalent thereto, and an L chain V region comprising CDRsincluded in an L chain V region having the amino acid sequence shown inSEQ ID NO: 12 or CDRs functionally equivalent thereto;

(b) an H chain V region comprising CDRs included in an H chain V regionhaving the amino acid sequence shown in SEQ ID NO: 20 or CDRsfunctionally equivalent thereto, and an L chain V region comprising CDRsincluded in an L chain V region having the amino acid sequence shown inSEQ ID NO: 28 or CDRs functionally equivalent thereto;

(c) an H chain V region comprising CDRs included in an H chain V regionhaving the amino acid sequence shown in SEQ ID NO: 36 or CDRsfunctionally equivalent thereto, and an L chain V region comprising CDRsincluded in an L chain V region having the amino acid sequence shown inSEQ ID NO: 44 or CDRs functionally equivalent thereto; and

(d) an H chain V region comprising CDRs included in an H chain V regionhaving the amino acid sequence shown in SEQ ID NOs: 68, 72, 76 or 80, orCDRs functionally equivalent thereto, and an L chain V region comprisingCDRs included in an L chain V region having the amino acid sequenceshown in SEQ ID NO 60 or CDRs functionally equivalent thereto, andwherein the antibody is capable of binding to a CDH3 polypeptide or apartial peptide thereof.

In preferred embodiments, CDRs may be determined by the Kabat definition(Kabat E. A. et al. (1991) Sequence of Proteins of ImmunologicalInterest. 5th Edition). The CDRs of each clone determined by the Kabatdefinition are described below.

The amino acid sequences of CDRs of clone #3 are (SEQ ID NO: 6)CDR1: SFWIH, (SEQ ID NO: 8) CDR2: NIDPSDSETHYNQYFKD and (SEQ ID NO: 10)CDR3: GGTGFSS in the H chain V region, and (SEQ ID NO: 14)CDR1: KASQDIDSYLS, (SEQ ID NO: 16) CDR2: RANRLVD, and (SEQ ID NO: 18)CDR3: LQYDEFPRT in the L chain V region.The amino acid sequences of CDRs of clone #4 are (SEQ ID NO: 21)CDR1: SYWMH, (SEQ ID NO: 24) CDR2: NIDPSDSETHYNQNFND and (SEQ ID NO: 26)CDR3: GGTGFAY in the H chain V region, and (SEQ ID NO: 30)CDR1: KASQDINNYLG, (SEQ ID NO: 32) CDR2: RTDRLIE, and (SEQ ID NO: 34)CDR3: LQYDEFPRM in the L chain V region.The amino acid sequences of CDRs of clone #5 are (SEQ ID NO: 38)CDR1: SYWMH, (SEQ ID NO: 40) CDR2: NIDPSDSETHYNQKFNDRA and(SEQ ID NO: 42) CDR3: GGTGFAY in the H chain V region, and(SEQ ID NO: 46) CDR1: KASQDINNYLG, (SEQ ID NO: 48) CDR2: RTDRLIE, and(SEQ ID NO: 50) CDR3: LQYDEFPRM in the L chain V region.The amino acid sequences of CDRs of variants of clone #6 are(SEQ ID NO: 54) CDR1: SYWIH, (SEQ ID NO: 70) CDR2: EIDPSDSYTYYNQNFKG,(SEQ ID NO: 74) EIDPSDTY-TYYNQNFKG, (SEQ ID NO: 78) EIDPSDAYTYYNQNFKG or(SEQ ID NO: 82) EIDPSDQYTYYNQNFKG and (SEQ ID NO: 58) CDR3: SGYGNLFVYin the H chain V region, and (SEQ ID NO: 62) CDR1: SATSSVTYMY,(SEQ ID NO: 64) CDR2: RTSNLAS, and (SEQ ID NO: 66) CDR3: QHYHIYPRTin the L chain V region.

Therefore, the present invention also provides antibodies or fragmentsthereof, wherein the antibodies comprises an H chain V region and an Lchain V region, wherein the H chain V region and the L chain V regionselected from the group consisting of:

(a) an H chain V region comprising CDR1 having the amino acid sequenceshown in SEQ ID NO: 6, CDR2 having the amino acid sequence shown in SEQID NO: 8 and CDR3 having the amino acid sequence shown in SEQ ID NO: 10,and an L chain V region comprising CDR1 having the amino acid sequenceshown in SEQ ID NO: 14, CDR2 having the amino acid sequence shown in SEQID NO: 16 and CDR3 having the amino acid sequence shown in SEQ ID NO:18;

(b) an H chain V region comprising CDR1 having the amino acid sequenceshown in SEQ ID NO: 22, CDR2 having the amino acid sequence shown in SEQID NO: 24 and CDR3 having the amino acid sequence shown in SEQ ID NO:26, and an L chain V region comprising CDR1 having the amino acidsequence shown in SEQ ID NO: 30, CDR2 having the amino acid sequenceshown in SEQ ID NO: 32 and CDR3 having the amino acid sequence shown inSEQ ID NO: 34;

(c) an H chain V region comprising CDR1 having the amino acid sequenceshown in SEQ ID NO: 38, CDR2 having the amino acid sequence shown in SEQID NO: 40 and CDR3 having the amino acid sequence shown in SEQ ID NO:42, and an L chain V region comprising CDR1 having the amino acidsequence shown in SEQ ID NO: 46, CDR2 having the amino acid sequenceshown in SEQ ID NO: 48 and CDR3 having the amino acid sequence shown inSEQ ID NO: 50; and

(d) an H chain V region comprising CDR1 having the amino acid sequenceshown in SEQ ID NO: 54, CDR2 having the amino acid sequence shown in SEQID NOs: 70, 74, 78 or 82 and CDR3 having the amino acid sequence shownin SEQ ID NO: 58, and an L chain V region comprising CDR1 having theamino acid sequence shown in SEQ ID NO: 62, CDR2 having the amino acidsequence shown in SEQ ID NO: 64 and CDR3 having the amino acid sequenceshown in SEQ ID NO: 66.

An example of the H chain V region (VH) in the above-mentioned “H chainV region comprising CDR1 having the amino acid sequence shown in SEQ IDNO: 6, CDR2 having the amino acid sequence shown in SEQ ID NO: 8 andCDR3 having the amino acid sequence shown in SEQ ID NO: 10” is a VHhaving the amino acid sequence shown in SEQ ID NO: 4. An example of theL chain V region (VL) in the above-mentioned “L chain V regioncomprising CDR1 having the amino acid sequence shown in SEQ ID NO: 14,CDR2 having the amino acid sequence shown in SEQ ID NO: 16 and CDR3having the amino acid sequence shown in SEQ ID NO: 8” is a VL having theamino acid sequence shown in SEQ ID NO: 12.

An example of the VH in the above-mentioned “H chain V region comprisingCDR1 having the amino acid sequence shown in SEQ ID NO: 22, CDR2 havingthe amino acid sequence shown in SEQ ID NO: 24 and CDR3 having the aminoacid sequence shown in SEQ ID NO: 26” is a VH having the amino acidsequence shown in SEQ ID NO: 20. An example of the VL in theabove-mentioned “L chain V region comprising CDR1 having the amino acidsequence shown in SEQ ID NO: 30, CDR2 having the amino acid sequenceshown in SEQ ID NO: 32 and CDR3 having the amino acid sequence shown inSEQ ID NO: 34” is a VL having the amino acid sequence shown in SEQ IDNO: 28.

An example of the VH in the above-mentioned “H chain V region comprisingCDR1 having the amino acid sequence shown in SEQ ID NO: 38, CDR2 havingthe amino acid sequence shown in SEQ ID NO: 40 and CDR3 having the aminoacid sequence shown in SEQ ID NO: 42” is a VH having the amino acidsequence shown in SEQ ID NO: 36. An example of the VL in theabove-mentioned “L chain V region comprising CDR1 having the amino acidsequence shown in SEQ ID NO: 46, CDR2 having the amino acid sequenceshown in SEQ ID NO: 48 and CDR3 having the amino acid sequence shown inSEQ ID NO: 50” is a VL having the amino acid sequence shown in SEQ IDNO: 44.

An example of the VH in the above-mentioned “H chain V region comprisingCDR1 having the amino acid sequence shown in SEQ ID NO: 54, CDR2 havingthe amino acid sequence shown in SEQ ID NOs: 70, 74, 78 or 82 and CDR3having the amino acid sequence shown in SEQ ID NO: 58” is a VH havingthe amino acid sequence shown in SEQ ID NO: 68, 72, 76 or 80. An exampleof the VL in the above-mentioned “L chain V region comprising CDR1having the amino acid sequence shown in SEQ ID NO: 62, CDR2 having theamino acid sequence shown in SEQ ID NO: 64 and CDR3 having the aminoacid sequence shown in SEQ ID NO: 66” is a VL having the amino acidsequence shown in SEQ ID NO: 60.

Therefore, in an embodiment, the present invention provides theantibodies or fragments thereof, wherein the antibody comprises an Hchain V region having the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4, 20, 36, 68, 72, 76 and 80 and/or an L chainV region having the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 12, 28, 44 and 60.

In preferred embodiments, the antibodies of the present inventioncomprises:

(a) an H chain V region having the amino acid sequence shown in SEQ IDNO: 4 and an L chain V region having the amino acid sequence shown inSEQ ID NO: 12;

(b) an H chain V region having the amino acid sequence shown in SEQ IDNO: 20 and an L chain V region having the amino acid sequence shown inSEQ ID NO: 28;

(c) an H chain V region having the amino acid sequence shown in SEQ IDNO: 36 and an L chain V region having the amino acid sequence shown inSEQ ID NO: 44; or

(d) an H chain V region having the amino acid sequence shown in SEQ IDNOs: 68, 72, 76 or 80 and an L chain V region having the amino acidsequence shown in SEQ ID NO: 60.

The antibodies of the present invention can be prepared by conventionalmethods. For example, the antibodies may be prepared by integrating apolypeptide encoding the antibody polypeptide into a suitable vector,introducing the vector into a host, and producing the antibody from thehost according to a conventional genetic recombination technique (see,for example, Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192,767-75).

The nucleic acid sequences of the polynucleotides encoding the V regionsof the antibodies of the present invention can be deduced from the aminoacid sequences of the V regions of the antibodies of the presentinvention. For example, the nucleic acid sequences shown in SEQ ID NOs:3 and 11 may be used as the nucleic acid sequences encoding the VH andVL of clone #3, respectively. For example, the nucleic acid sequencesshown in SEQ ID NOs: 19 and 27 may be used as the nucleic acid sequencesencoding the VH and VL of clone #4, respectively. For example, thenucleic acid sequences shown in SEQ ID NOs: 35 and 43 may be used as thenucleic acid sequences encoding the VH and VL of clone #5, respectively.For example, the nucleic acid sequences shown in SEQ ID NOs: 67, 71, 75or 79, and 59 may be used as the nucleic acid sequences encoding the VHand VL of variants of clone #6, respectively. The polynucleotidesencoding the V region of the antibodies of the present invention can besynthesized based on the sequence information by conventional methodssuch as the solid phase techniques (Beaucage S L & Iyer R P, Tetrahedron(1992) 48, 2223-311; Matthes et al., EMBO J. (1984) 3, 801-5) andoligonucleotide synthesis techniques (Jones et al. Nature (1986) 321,522-5).

The polynucleotide encoding the antibody V region are integrated into anexpression vector containing polynucleotide encoding the antibodyconstant (C) region. For the production of the antibody used in thepresent invention, the polypeptide encoding the antibody (antibody gene)is integrated into an expression vector so that the antibody gene can beexpressed under the control of expression control elements (e.g.,enhancer, promoter). A host cell is transformed with the expressionvector to express the antibody.

In the expression of the antibody gene, the polynucleotide encoding Hchain and polynucleotide encoding L chain of the antibody may beintegrated into separate expression vectors, and then a host cell isco-transformed with the resultant recombinant expression vectors.Alternatively, both polynucleotide encoding H chain and polynucleotideencoding L chain of the antibody may be integrated together into asingle expression vector, and then a host cell is transformed with theresultant recombinant expression vector (for example, WO 94/11523).

The antibody gene can be expressed by known methods. For the expressionin a mammalian cell, a conventional useful promoter, the antibody geneto be expressed and a poly(A) signal (located downstream to the 3′ endof the antibody gene) may be operably linked. For example, as the usefulpromoter/enhancer system, a human cytomegalovirus immediate earlypromoter/enhancer system may be used.

Other promoter/enhancer systems, for example, those derived from viruses(e.g., retrovirus, polyoma virus, adenovirus and simian virus 40 (SV40))and those derived from mammalian cells (e.g., human elongation factor 1alpha (HEF1 alpha)), may also be used for the expression of the antibodyin the present invention.

When SV40 promoter/enhancer system is used, the gene expression may beperformed readily by the method of Mulligan et al. (Nature (1979) 277,108-14.). When HEF1 alpha promoter/enhancer system is used, the geneexpression may be performed readily by the method of Mizushima et al.(Nucleic Acids Res. (1990) 18, 5322.).

For the expression in E. coli, a conventional useful promoter, a signalsequence for secreting the antibody of interest and the antibody genemay be operably linked. As the promoter, lacZ promoter or araB promotermay be used. When lacZ promoter is used, the gene expression may beperformed by the method of Ward et al. (Nature (1098) 341, 544-6.; FASBEJ. (1992) 6, 2422-7.), while when araB promoter is used, the geneexpression may be performed by the method of Better et al. (Science(1988) 240, 1041-3.).

With respect to the signal sequence for secretion of the antibody, whenthe antibody of interest is intended to be secreted in a periplasmicspace of the E. coli, pelB signal sequence (Lei, S. P. et al, J.Bacteriol. (1987) 169, 4379-83.) may be used. The antibody secreted intothe periplasmic space is isolated and then refolded so that the antibodytakes an appropriate configuration.

The replication origin derived from viruses {e.g., SV40, polyoma virus,adenovirus, bovine papilloma virus (BPV)) or the like may be used. Inorder to increase the gene copy number in the host cell system, theexpression vector may further contain a selective marker gene, such asan aminoglycoside phosphotranferase (APH) gene, a thymidine kinase (TK)gene, an E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt)gene and a dihydrofolate reductase (dhfr) gene. For the production ofthe antibody used in the present invention, any expression systemincluding eukaryotic and prokaryotic cell systems may be used. Theeukaryotic cell includes established cell lines of animals (e.g.,mammals, insects, molds and fungi, yeast). The prokaryotic cell includesbacterial cells such as E. coli cells. It is preferable that theantibody used in the present invention be expressed in a mammalian cell,such as a CHO, COS, myeloma, BHK, Vero and HeLa cell.

Next, the transformed host cell is cultured in vitro or in vivo toproduce the antibody of interest. The cultivation of the host cell maybe performed by any known method. The culture medium that can be usedherein may be DMEM, MEM, RPMI 1640 or IMDM medium. The culture mediummay contain a serum supplement, such as fetal calf serum (FCS).

In the production of the recombinant antibody, besides theabove-mentioned host cells, a transgenic animal may also be used as ahost. For example, the antibody gene is inserted into a predeterminedsite of a gene encoding a protein inherently produced in the milk of ananimal (e.g., beta-casein) to prepare a fusion gene. A DNA fragmentcontaining the antibody gene-introduced fusion gene is injected into anembryo of a non-human animal, and the embryo is then introduced into afemale animal. The female animal having the embryo therein bears atransgenic non-human animal. The antibody of interest is secreted in themilk from the transgenic non-human animal or a progeny thereof. For thepurpose of increasing the amount of the antibody-containing milk, anappropriate hormone may be administered to the transgenic animal (Ebert,K. M. et al, Bio/Technology (1994) 12, 699-702.).

The antibody expressed and produced as described above may be isolatedfrom the cells or the host animal body and purified. The isolation andpurification of the antibody used in the present invention may beperformed on an affinity column. Other methods conventionally used forthe isolation and purification of an antibody may be also be used; thusthe method is not particularly limited. For example, variouschromatographies, filtration, ultrafiltration, salting out and dialysismay be used singly or in combination to isolate and purify the antibodyof interest (Antibodies A Laboratory Manual. Ed. Harlow, David Lane,Cold Spring Harbor Laboratory, 1988).

(ii) Chimeric Antibody and Humanized Antibody

In the present invention, an artificially modified recombinant antibodymay be used, including a chimeric antibody and a humanized antibody.These modified antibodies can be prepared by any known method. Forexample, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. ScL, (1984) 81: 6851-5.;Neuberger et al., Nature (1984), 312: 604-8.; Takeda et al., Nature(1985) 314: 452-4.) can be used.

A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine monoclonal antibody and a humanimmunoglobulin constant region, e.g., “humanized antibodies”.

A chimeric antibody according to the present invention can be preparedby ligating the DNA encoding the antibody V region to DNA encoding ahuman antibody C region, integrating the ligation product into anexpression vector, and introducing the resultant recombinant expressionvector into a host to produce the chimeric antibody.

A humanized antibody is also referred to as “reshaped human antibody”,in which the CDRs of an antibody of a non-human mammal (e.g., a mouse)are grafted to those of a human antibody. The general geneticrecombination procedure for producing such humanized antibody is alsoknown (for example, EP 125023; WO 96/02576.).

Specifically, a nucleic acid sequence in which mouse antibody CDRs areligated through framework regions (FRs) is designed, and synthesized bya PCR method using several oligonucleotides as primers which weredesigned to have regions overlapping to the terminal regions of the CDRsand the FRs. The resultant DNA is ligated to DNA encoding the humanantibody C-region, and the ligation product is integrated into anexpression vector. The resultant recombinant expression vector isintroduced into a host, thereby producing the humanized antibody (forexample, WO 96/02576).

The FRs ligated through the CDRs are selected so that the CDRs can forma functional antigen binding site. If necessary, an amino acid(s) in theFRs of the antibody V region may be replaced so that the CDRs of thereshaped human antibody can form an appropriate antigen binding site(Sato, K. et al., Cancer Res. (1993) 53, 851-6.).

The chimeric antibody is composed of V regions derived from a non-humanmammal antibody and C-regions derived from a human antibody. Thehumanized antibody is composed of CDRs derived from a non-human mammalantibody and FRs and C regions derived from a human antibody. Thehumanized antibody may be useful for clinical use, because theantigenicity of the antibody against a human body is reduced.

A specific example of a chimeric antibody or a humanized antibody usedin the present invention is an antibody in which the V regions arederived from the mouse monoclonal antibody clone #3, #4, #5 or variantsof clone #6, or an antibody in which the CDRs are derived from the mousemonoclonal antibody clone #3, #4, #5 or variants of clone #6. The methodfor producing such chimeric antibodies and humanized antibodies aredescribed below.

For example, first, the genes encoding the V regions or CDRs of theantibodies of clone #3, #4, #5 or variants of clone #6 are prepared fromthe RNAs of antibody-producing cells by polymerase chain reaction (PCR)or such (see, for example, Larrick et al., “Methods: a Companion toMethods in Enzymology”, Vol. 2: 106, 1991; Courtenay-Luck, “GeneticManipulation of Monoclonal Antibodies” in Monoclonal Antibodies:Production, Engineering and Clinical Application; Ritter et al. (eds.),page 166, Cambridge University Press, 1995, and Ward et al., “GeneticManipulation and Expression of Antibodies” in Monoclonal Antibodies:Principles and Applications; and Birch et al. (eds.), page 137,Wiley-Liss, Inc., 1995). The polynucleotides encoding the V regions orCDRs of the antibodies may be synthesized oligonucleotide synthesistechniques (e.g., Jones et al. Nature (1986) 321, 522-5). Then, theamplified or synthesized products are subjected to agarose gelelectrophoresis according to conventional procedures, and DNA fragmentsof interest are excised, recovered, purified and ligated to a vectorDNA.

The obtained DNA and vector DNA can be ligated using a known ligationkit to construct a recombinant vector. A vector DNA may be prepared in aknown method: J. Sambrook, et al., “Molecular Cloning”, Cold SpringHarbor Laboratory Press, 1989. The vector DNA is digested withrestriction enzyme(s), and the nucleotide sequence of a desired DNA canbe determined by a known method or using an automated sequencer.

Once DNA fragments encoding for L and H chain V regions of mousemonoclonal antibody (hereinafter L or H chain of an antibody maysometimes be referred to as “mouse L or H chain” for mouse antibodiesand “human L or H chain” for human antibodies) are cloned, the DNAsencoding mouse V regions and DNAs encoding human antibody C regions areligated and expressed to yield chimeric antibodies.

A standard method for preparing chimeric antibodies involves ligating amouse signal sequence and V region sequence present in a cloned cDNA toa sequence encoding for a human antibody C region already present in anexpression vector of a mammalian cell. Alternatively, a mouse signalsequence and V region sequence present in a cloned cDNA are ligated to asequence coding for a human antibody C region followed by ligation to amammalian cell expression vector.

The polypeptide comprising human antibody C region can be any of H or Lchain C regions of human antibodies, including, for example, C gamma 1,C gamma 2, C gamma 3 or C gamma 4 for human H chains or C lambda or Ckappa for L chains. To prepare a chimeric antibody, two expressionvectors are first constructed; that is, an expression vector containingDNAs encoding mouse L chain V region and human L chain C region underthe control of an expression control element such as anenhancer/promoter system, and an expression vector containing DNAsencoding mouse H chain V region and human H chain C region under thecontrol of an expression control element such as an enhancer/promotersystem, are constructed. Then, host cells such as mammalian cells (forexample, COS cell) are cotransformed with these expression vectors andthe transformed cells are cultivated in vitro or in vivo to produce achimeric antibody (see, for example, WO91/16928).

Alternatively, the mouse signal sequence present in the cloned cDNA andDNAs encoding mouse L chain V region and human L chain C region as wellas the mouse signal sequence and DNAs coding for mouse H chain V regionand human H chain C region are introduced into a single expressionvector (see, for example, WO94/11523) and said vector is used totransform a host cell; then, the transformed host is cultured in vivo orin vitro to produce a desired chimeric antibody. The vector for theexpression of H chain of a chimeric antibody can be obtained byintroducing cDNA comprising a nucleotide sequence encoding mouse H chainV region (hereinafter referred to also as “cDNA for H chain V region”)into a suitable expression vector containing the genomic DNA comprisinga nucleic acid sequence encoding H chain C region of human antibody(hereinafter referred to also as “genomic DNA for H chain C region”) orcDNA encoding the region (hereinafter referred to also as “cDNA for Hchain C region”). The H chain C region includes, for example, C gamma 1,C gamma 2, C gamma 3 or C gamma 4 regions.

The expression vectors having the genomic DNA coding for H chain Cregion, in particular, those encoding C gamma 1 region, include, forexample, HEF-PMh-g gamma 1 (WO92/19759) and DHER-INCREMENT E-RVh-PM1-f(WO92/19759). Alternatively, human C region library can be preparedusing cDNA from human PBMC (peripheral blood mononuclear cells) asdescribed previously (Liu, A. Y. et al., Proc. Natl. Acad. Sci. USA,Vol. 84, 3439-43, 1987; Reff, M. E. et al., Blood, Vol. 83, No. 2,435-45, 1994). When cDNA encoding mouse H chain V region is insertedinto these expression vectors, an appropriate nucleic acid sequence canbe introduced into the cDNA through PCR method. For instance, PCR may beeffected using a PCR primer which is designed such that the cDNA has arecognition sequence for a suitable restriction enzyme at its 5′-end andKozak consensus sequence immediately before the initiation codon thereofso as to improve the transcription efficiency, as well as a PCR primerwhich is designed such that said cDNA has a recognition sequence for asuitable restriction enzyme at its 3′-end and a splice donor site forproperly splicing the primary transcription products of the genomic DNAto give a mRNA, to introduce these appropriate nucleic acid sequencesinto the expression vector.

The constructed cDNA encoding mouse H chain V region may be treated witha suitable restriction enzyme(s), then it is inserted into theexpression vector to construct a chimeric H chain expression vectorcontaining the genome DNA encoding H chain C region (C gamma 1 region).Alternatively, the constructed cDNA encoding mouse H chain V region maybe treated with a suitable restriction enzyme(s), ligated to cDNA codingfor the H chain C region C gamma 1, and inserted into an expressionvector such as pQCXIH (Clontech) to construct an expression vectorcontaining the cDNA encoding a chimeric H chain.

The vector for the expression of L chain of a chimeric antibody can beobtained by ligating a cDNA coding for mouse L chain V region and agenomic DNA or cDNA coding for L chain C region of a human antibody andintroducing into a suitable expression vector. The L chain C regionincludes, for example, kappa chain and lambda chain. When an expressionvector containing cDNA encoding mouse L chain V region is constructed,appropriate nucleic acid sequences such as a recognition sequence orKozak consensus sequence can be introduced into said expression vectorthrough PCR method.

The entire nucleic acid sequence of cDNA encoding human L lambda chain Cregion may be synthesized by a DNA synthesizer and constructed throughPCR method. The human L lambda chain C region is known to have at least4 different isotypes and each isotype can be used to construct anexpression vector. The constructed cDNA encoding human L lambda chain Cregion and the above constructed cDNA encoding mouse L chain V regioncan be ligated between suitable restriction enzyme sites and insertedinto an expression vector such as pQCXIH (Clontech), to construct anexpression vector containing cDNA coding for a L lambda chain of achimeric antibody. The DNA encoding human L kappa chain C region to beligated to the DNA coding for mouse L chain V region can be constructedfrom, for example, HEF-PM1 k-gk containing the genomic DNA (seeWO92/19759). Alternatively, human C region library can be prepared usingcDNA from human PBMC (peripheral blood mononuclear cells) as describedpreviously (Liu, A. Y. et al., Proc. Natl. Acad. Sci. USA, Vol. 84,3439-43, 1987; Reff, M. E. et al., Blood, Vol. 83, No. 2, 435-45, 1994).

Recognition sequences for suitable restriction enzymes can beintroduced, through PCR method, into 5′- and 3′-ends of DNA coding for Lkappa chain C region, and the DNA coding for mouse L chain V region asconstructed above and the DNA encoding L kappa chain C region can beligated to each other and inserted into an expression vector such aspQCXIH (Clontech) to construct an expression vector containing cDNAencoding L kappa chain of a chimeric antibody.

Humanization of non-human antibodies can be essentially performedfollowing the method of Winter and co-workers (Jones et al., Nature,321: 522-525 (1986); Reichmann et al., Nature, 332: 323-327 (1988);Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting CDRsequences for the corresponding sequences of a human antibody.

In order to make a humanized antibody in which CDR of a mouse monoclonalantibody is grafted to a human antibody, it is desirable that thereexists a high homology between FR of the mouse monoclonal antibody andFR of the human antibody. Accordingly, a comparison is made between Vregions of H and L chains of mouse monoclonal antibody clone #3, #4, #5or variants of clone #6, and the V regions of all the known antibodieswhose structures have been elucidated with the use of Protein Data Bank.Further, they are simultaneously compared with the human antibodysubgroups (HSG: Human subgroup) classified by Kabat et al. based on thelength of antibody FR, the homology of amino acids, and the like (Kabat,E′. A. et al, US Dep, Health and Human Services, US Government PrintingOffices, 1991).

The first step for designing DNA encoding a humanized antibody V regionis to select a human antibody V region as a basis for the designing. Forexample, FR of a human antibody V region having a homology of higherthan 80% with FR of a mouse antibody V region can be used in theproduction of a humanized antibody.

In the humanized antibody, the C region and the framework (FR) regionsof the V region of the antibody are originated from human and the CDRsof the V region are originated from mouse. A polypeptide comprising theV region of the humanized antibody can be produced in the manner calledCDR-grafting by PCR method so long as a DNA fragment of a human antibodywould be available as a template. The “CDR-grafting” refers to a methodwherein a DNA fragment encoding a mouse-derived CDRs are made andreplaced for the CDRs of a human antibody as a template. If a DNAfragment of a human antibody to be used as a template is not available,a nucleic acid sequence registered in a database may be synthesized in aDNA synthesizer and a DNA for a V region of a humanized antibody can beproduced by the PCR method. Further, when only an amino acid sequence isregistered in the database, the entire nucleic acid sequence may bededuced from the amino acid sequence on the basis of knowledge on thecodon usage in antibodies as reported by Kabat, E. A. et al. in US Dep.Health and Human Services, US Government Printing Offices, 1991. Thisnucleic acid sequence is synthesized in a DNA synthesizer and a DNA of ahumanized antibody V region can be prepared by PCR method and introducedinto a suitable host followed by expression thereof to produce thedesired polypeptide. General procedures of CDR-grafting by PCR methodare described below when a DNA fragment of a human antibody as atemplate is available.

First, mouse derived DNA fragments corresponding to respective CDRs aresynthesized. CDRs 1 to 3 are synthesized on the basis of the nucleicacid sequences of the previously cloned mouse H and L chain V regions.For example, when a humanized antibody is produced based on the mousemonoclonal antibody clone #3, CDR sequences of H chain V region can bethe amino acid sequences as shown in SEQ ID NOs: 6 (VH CDR1), 8 (VHCDR2) and 10 (VH CDR3); and CDR sequences of L chain V region can be theamino acid sequences as shown in SEQ ID NOs: 14 (VL CDR1), 16 (VL CDR2)and 18 (VL CDR3). When a humanized antibody is produced based on themouse monoclonal antibody clone #4, CDR sequences of H chain V regioncan be the amino acid sequences as shown in SEQ ID NOs: 22 (VH CDR1), 24(VH CDR2) and 26 (VH CDR3); and CDR sequences of L chain V region can bethe amino acid sequences as shown in SEQ ID NOs: 30 (VL CDR1), 32 (VLCDR2) and 34 (VL CDR3). When a humanized antibody is produced based onthe mouse monoclonal antibody clone #5, CDR sequences of H chain Vregion can be the amino acid sequences as shown in SEQ ID NOs: 38 (VHCDR1), 40 (VH CDR2) and 42 (VH CDR3); and CDR sequences of L chain Vregion can be the amino acid sequences as shown in SEQ ID NOs: 46 (VLCDR1), 48 (VL CDR2) and 50 (VL CDR3). When a humanized antibody isproduced based on the mouse monoclonal antibody variants of clone #6,CDR sequences of H chain V region can be the amino acid sequences asshown in SEQ ID NOs: 54 (VH CDR1), 70, 74, 78, or 82 (VH CDR2) and 66(VH CDR3); and CDR sequences of L chain V region can be the amino acidsequences as shown in SEQ ID NOs: 62 (VL CDR1), 64 (VL CDR2) and 66 (VLCDR3).

The DNA for H chain V region of a humanized antibody may be ligated toDNA for any human antibody H chain C region, for example, human H chainC gamma 1 region. As mentioned above, the DNA for H chain V region maybe treated with a suitable restriction enzyme and ligated to a DNAencoding a human H chain C region under an expression control elementsuch as an enhancer/promoter system to make an expression vectorcontaining DNAs for a humanized H chain V region and a human H chain Cregion.

The DNA for L chain V region of a humanized antibody may be ligated toDNA for any human antibody L chain C region, for example, human L chainC lambda region. The DNA for L chain V region may be treated with asuitable restriction enzyme and ligated to a DNA encoding a human Llambda chain C region under an expression control element such as anenhancer/promoter system to make an expression vector containing DNAsencoding a humanized L chain V region and a human L lambda chain Cregion.

The DNA encoding H chain V region of a humanized antibody and a human Hchain C region and the DNA encoding a humanized L chain V region andhuman L chain C region may also be introduced into a single expressionvector such as that disclosed in WO94/11523, said vector may be used totransform a host cell, and the transformed host may be cultivated invivo or in vitro to produce a desired humanized antibody.

To produce a chimeric or humanized antibody, two expression vectors asabove mentioned should be prepared. Thus, with respect to a chimericantibody, an expression vector comprising a DNA coding for a mouse Hchain V region and a human H chain C region under the control of anexpression control element such as an enhancer/promoter, and anexpression vector comprising a DNA coding for a mouse L chain V regionand a human L chain C region under the control of an expression controlelement are constructed. With respect to a humanized antibody, anexpression vector comprising a DNA encoding a humanized H chain V regionand a human H chain C region under the control of an expression controlelement, and an expression vector comprising a DNA encoding a humanizedL chain V region and a human L chain C region under the control of anexpression control element are constructed.

Then, a host cell such as a mammalian cell (for example, COS cell) maybe cotransformed with these expression vectors and the resultingtransformed cell may be cultured in vitro or in vivo to produce thechimeric or humanized antibody (see, for example, WO91/16928).Alternatively, a DNA coding for H chain V and C regions and a DNAencoding L chain V and C regions may be ligated to a single vector andtransformed into a suitable host cell to produce an antibody. Thus, inthe expression of a chimeric antibody, a DNA coding for a mouse leadersequence present in the cloned cDNA, a mouse H chain V region and ahuman H chain C region as well as a DNA coding for a mouse leadersequence, a mouse L chain V region and a human L chain C region, can beintroduced into a single expression vector such as one disclosed in e.g.WO94/11523. In the expression of a humanized antibody, a DNA encoding ahumanized H chain V region and a human H chain C region and a DNA codingfor a humanized L chain V region and a human L chain C region may beintroduced into a single expression vector such as one disclosed in e.g.WO94/11523. Such a vector is used to transform a host cell and thetransformed host is cultured in vivo or in vitro to produce a chimericor humanized antibody of interest.

Any expression system may be used to produce the chimeric or humanizedantibodies of the present invention. For example, eukaryotic cellsinclude animal cells such as established mammalian cell lines, fungalcells, and yeast cells; prokaryotic cells include bacterial cells suchas Escherichia coli. Preferably, the chimeric or humanized antibody ofthe present invention is expressed in a mammalian cell such as COS orCHO cell.

Any conventional promoters useful for the expression in mammalian cellsmay be used. For example, human cytomegalovirus (HCMV) immediate earlypromoter is preferably used. In addition, promoters for gene expressionin mammalian cells may include virus promoters, such as those ofretrovirus, polyoma virus, adenovirus and simian virus (SV) 40, andmammalian cell derived promoters, such as those of human polypeptidechain elongation factor-1 alpha (HEF-I alpha). For example, SV40promoter may be readily used according to Mulligan et al. method(Nature, 277, 108-14, 1979); Mizushima, S. et al. method (Nucleic AcidsResearch, 18, 5322, 1990) may be easily used with HEF-1 alpha promoter.

Replication origin includes those derived from SV40, polyoma virus,adenovirus or bovine papilloma virus (BPV). Further, the expressionvector may comprise a gene for phosphotransferase APH(3′) II or I (neo),thymidine kinase (TK), E. coli xanthineguanine phosphoribosyltransferase(Ecogpt) or dihydrofolate reductase (DHFR) as a selective marker forincreasing the gene copy number in a host cell system.

The chimeric or humanized antibody of interest which is thus produced byculturing the transformant transformed with a DNA coding for thechimeric or humanized antibody may be isolated from the cell and thenpurified.

The isolation and purification of the chimeric or humanized antibody ofinterest may be carried out by using a protein A agarose column, but mayalso be performed by any methods used in isolation and purification of aprotein and thus is not limited. For instance, a chromatography,ultrafiltration, salting out and dialysis may optionally be selected orcombined to isolate and purify the chimeric or humanized antibody.

After isolating the chimeric antibody or humanized antibody, theconcentration of the resulting purified antibody can be determined byELISA.

The determination of the antigen-binding activity or other activitiesincluding binding activity to a normal cell of the chimeric antibody orhumanized antibody may be performed by any known methods (Antibodies ALaboratory Manual, Ed. Harlow, David Lane, Cold Spring HarborLaboratory, 1988). As the method for the determination of theantigen-binding activity of an antibody, techniques such as ELISA(enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA(radioimmunoassay) or fluorescent assay may be employed. In ELISA, anantibody is immobilized on a plate, and an antigen for the antibody isadded to the plate, then a sample containing the desired antibody, suchas the culture supernatant of antibody-producing cells or a purifiedantibody is added. Next, a secondary antibody which recognizes theprimary antibody and is tagged with an enzyme such as alkalinephosphatase is added to the plate, and this is preincubated. Afterwashing, an enzyme substrate such as p-nitrophenyl phosphate is added tothe plate, and the absorbance is measured to evaluate theantigen-binding ability of the sample of interest. The evaluation ofantigen-binding activity may also be performed using BIAcore(Pharmacia).

To retain the antigen-binding activity, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen, is achieved.

(iii) Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24: 107-117 (1992) and Brennan etal., Science, 229: 81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F (ab′) 2 fragments (Carter etal., Bio/Technology 10: 163-167 (1992)). According to another approach,F (ab′) 2 fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. Theantibody fragment may also be a “linear antibody”, e.g., as described inU.S. Pat. No. 5,641,870 for example. Such linear antibody fragments maybe monospecific or bispecific.

(iv) Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary, an anti-cancer cell markerbinding arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 orCD3), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII(CD32) and FcyRIH (CD 16) so as to focus cellular defense mechanisms tothe cancer cell. Bispecific antibodies may also be used to localizecytotoxic agents to the cancer cell. These antibodies possess a cancercell marker-binding arm and an arm which binds the cytotoxic agent (e.g.saporin, anti-interferon-a, vinca alkaloid, ricin A chain, methotrexateor radioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F (ab) 2 bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305: 537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10: 3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CHI) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121: 210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 domain of an antibody constant domain. In this method,one or more small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g. tyrosine ortryptophan). Compensatory “cavities” of identical or similar size to thelarge side chains) are created on the interface of the second antibodymolecule by replacing large amino acid side chains with smaller ones(e.g. alanine or threonine). This provides a mechanism for increasingthe yield of the heterodimer over other unwanted end-products such ashomodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F (ab′) 2 fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 2 17-225 (1992) describethe production of a fully humanized bispecific antibody F (ab′) 2molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers (Kostelny et al., J Immunol. 148 (5): 1547-1553 (1992)).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90: 6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J Immunol., 152: 5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J; Immunol. 147: 60(1991).

(v) Antibody Conjugates and Other Modifications

The antibodies of the present invention are optionally conjugated to acytotoxic or therapeutic agent.

For example, a therapeutic agent includes any chemotherapeutic agentwhich is useful in the treatment of cancer. Examples of chemotherapeuticagents include alkylating agents such as thiotepa and cyclosphosphamide;alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembiehin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromoinycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idambicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, poffiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK&commat;razoxane; sizofrran; spirogermanium; tenuazonic acid; triaziquone; 2,2′, 2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOLO, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTEW, Rh6ne-Poulenc Rorer, Antony, France); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also, therapeutic agents includeanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4 (5)-imidazoles, 4 hydroxytamoxifen,trioxifene, keoxifene, onapristone, and toremifene (Fareston); andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, a maytansine (U.S. Pat. No. 5,208,020), a trichothene,and CC 1065 are also contemplated herein. In one preferred embodiment ofthe invention, the antibodies are conjugated to one or more maytansinemolecules (e.g. about 1 to about 10 maytansine molecules per antibodiesmolecule). Maytansine may, for example, be converted to May SS-Me whichmay be reduced to May-SH3 and reacted with modified antibodies (Chari etal. Cancer Research 52: 127-131 (1992)) to generate amaytansinoid-antibody conjugate.

Alternatively, the antibody may be conjugated to one or morecalicheamicin molecules. The calicheamicin family of antibiotics iscapable of producing double stranded DNA breaks at sub-picomolarconcentrations. Structural analogues of calicheamicin which may be usedinclude, but are not limited to gammall, alpha2I, alpha3I,N-acetyl-gammall, PSAG and OI1 (Hinman et al. Cancer Research 53:3336-3342 (1993) and Lode et al, Cancer Research 58: 2925-2928 (1998)).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates antibody conjugated with avariety of radioactive isotopes. Examples include ¹¹¹In, ²¹¹At, ¹³¹I,¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P and radioactive isotopes ofLu. In the present invention, the antibody of the present invention maybe labeled with radio-nuclides just prior to use, or provided asradiolabeled antibody. The skilled practitioner will realize that thereare numerous radionuclides and chemocytotoxic agents that can be coupledto tumor-specific antibodies by well-known techniques and delivered to asite to specifically damaging tumor cells and tissue. (See, for example,U.S. Pat. No. 4,542,225 to W. A. Blattler et al., issued Sep. 17, 1985;and Pastan et al., 1986, Cell, 47:641-648). For example, imaging andcytotoxic reagents that are suitable for use include ¹²⁵I, ¹²³I. ¹¹¹In(e.g., Sumerdon et al., 1990, Nucl. Med. Biol., 17:247-254), and 99 mTc;fluorescent labels such as fluorescein and rhodamine; chemiluminescentlabels such as luciferin, and paramagnetic ions for use in magneticresonance imaging (Lauffer et al., 1991, Magnetic Resonance in Medicine,22:339-342). Antibodies can be labeled with such reagents usingprotocols and techniques known and practiced in the art. See, forexample, Wenzel and Meares, Radioimmunoimaging and Radioimmunotherapy,Elsevier, New York, 1983; Colcer et al., 1986, Meth. Enzymol.,121:802-816; and Monoclonal Antibodies for Cancer Detection and Therapy,Eds. Baldwin et al., Academic Press, 1985, pp. 303-316, for techniquesrelating to the radiolabeling of antibodies. Yttrium-90 (90Y) labeledmonoclonal antibodies have been described for maximizing the dosedelivered to the tumor or cancer cells and/or tissue, while limitingtoxicity to normal tissues (e.g., Goodwin and Meares, 1997, CancerSupplement, 80:2675-2680). Other cytotoxic radionuclides including, butnot limited to, Iodine-131 (131I) and Rhenium-186 can also be used forlabeling monoclonal antibodies of the present invention. Among theradionuclides, Yttrium-90 (90Y) may be suitable for radioimmunotherapy,since Yttrium-90 (90Y) provides advantages over Iodine-131 (131I)because it delivers higher beta energy (2.3 MeV vs 0.61 MeV) to thetumor and has path length of 5 to 10 mm resulting in the improvedability to kill both targeted and neighboring cells, an advantageparticularly in bulky or poorly vascularized tumor. Thedetectable/detecting label used is selected according to the imagingmodality to be used. For example, radioactive labels, such as Indium-111(¹¹¹In), Technetium-99m (^(99m)Tc), or Iodine 131 (¹³¹I), can be usedfor planar scans or for single photon emission computed tomography(SPECT). Also, positron-emitting labels such as Fluorine-19 can be usedin positron emission tomography (PET). Paramagnetic ions, such asGadlinium(III) or Manganese(II) can be used in magnetic resonanceimaging (MRI). The monoclonal antibodies can also be labeled withradio-opaque labels for the visualization of cancer cells afterinjection, for example, by X-ray, CATscan, or MRI. In particular, forCDH3 relating disease (e.g. cancers), localization of the label withinthe cancers permits the determination of the spread of the disease. Theamount of label that is present and detectable within the cancersexpressing CDH3, for example, allows the determination of the presenceor absence of cancer or tumor in the subject to be diagnosed.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyriylditliol) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon-14-labeled 1 isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody (See WO94/11026). Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, dimethyl linker or disulfide-containinglinker (Charm et al. Cancer Research 52: 127-131 (1992)) may be used.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g. by recombinant techniques or peptide synthesis.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g. avidin) whichis conjugated to a cytotoxic agent (e.g. a radionucleotide).

The antibodies of the present invention may also be conjugated with aprodrug activating enzyme which converts a prodrug (e.g. a peptidylchemotherapeutic agent, see WO81/01145) to an active anti-cancer drug(See, for example, WO 88/07378 and U.S. Pat. No. 4,975,278).

The enzyme component of such conjugates includes any enzyme capable ofacting on a prodrug in such a way so as to covert it into its moreactive, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic5-fluorocytosine into theanti-cancer drug, fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as 13-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs;13-lactamase useful for converting drugs derivatized with 13-lactamsinto free drugs; and penicillin amidases, such as penicillin V amidaseor penicillin G amidase, useful for converting drugs derivatized attheir amine nitrogens with phenoxyacetyl or phenylacetyl groups,respectively, into free drugs. Alternatively, antibodies with enzymaticactivity, also known in the art as “abzymes”, can be used to convert theprodrugs of the invention into free active drugs (see, e.g., Massey,Nature 328: 457-458 (1987)). Antibody-abzyme conjugates can be preparedas described herein for delivery of the abzyme to a tumor cellpopulation.

The enzymes of the present invention can be covalently bound to theantibody by techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 312: 604-608 (1984)).

Other modifications of the antibody are contemplated herein. Forexample, the antibody may be linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol.

The antibodies disclosed herein may also be formulated as liposomes.Liposomes containing the antibody are prepared by methods known in theart, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA,82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030(1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 publishedOct. 23, 1997. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of an antibody of the present invention can beconjugated to the liposomes as described in Martin et al. J; Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome (SeeGabizon et al. ANational Cancer Inst. 81 (19) 1484 (1989)).

Amino acid sequence modifications of antibodies described herein arecontemplated. For example, it may be desirable to improve the bindingaffinity and/or other biological properties of the antibody. Amino acidsequence variants of the antibody are prepared by introducingappropriate nucleotide changes into the antibody encoding nucleic acid,or by peptide synthesis. Such modifications include, for example,deletions from, and/or insertions into and/or substitutions of, residueswithin the amino acid sequences of the antibody. Any combination ofdeletion, insertion, and substitution is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. The amino acid changes also may alter posttranslationalprocesses of the antibody, such as changing the number or position ofglycosylation sites.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody ofan enzyme, or a polypeptide which increases the serum half-life of theantibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by different residue. The sites of greatest interest forsubstitutional mutagenesis of antibody include the hypervariableregions, but FR alterations are also contemplated.

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain.

Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophiuic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bonds may be added to the antibody toimprove its stability (particularly where the antibody is a fragmentsuch as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariants selected for further development will have improved biologicalproperties relative to the parent antibody from which they aregenerated. A convenient way for generating such substitutional variantsis affinity maturation using phage display. Briefly, severalhypervariable region sites (e.g. 6-7 sites) are mutated to generate allpossible amino substitutions at each site. The antibody variants thusgenerated are displayed in a monovalent fashion from filamentous phageparticles as fusions to the gene III product of M13 packaged within eachparticle. The phage-displayed variants are then screened for theirbiological activity (e.g. binding affinity) as herein disclosed. Inorder to identify candidate hypervariable region sites for modification,alanine scanning mutagenesis can be performed to identify hypervariableregion residues contributing significantly to antigen binding.Alternatively, or in addition, it may be beneficial to analyze a crystalstructure of the antigen-antibody complex to identify contact pointsbetween the antibody and antigen. Such contact residues and neighboringresidues are candidates for substitution according to the techniqueselaborated herein. Once such variants are generated, the panel ofvariants is subjected to screening as described herein and antibodieswith superior properties in one or more relevant assays may be selectedfor further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to modify the antibodies used in the presentinvention to improve effector function, e.g. so as to enhanceantigen-dependent cell-mediated cyotoxicity (ADCC) and/or complementdependent cytotoxicity (CDC) of the antibody. This may be achieved byintroducing one or more amino acid substitutions in an Fc region of anantibody. Alternatively or additionally, cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992)and Shopes, B. J limmunol 148: 2918-2922 (1992).

Homodimeric antibodies with enhanced anti-tumor activity may also beprepared using heterobifunctional cross-linkers as described in Wolff etal. Cancer Research 53: 2560-2565 (1993). Alternatively, an antibody canbe engineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities (See Stevenson et al.Anti-CancerDrugDesign 3: 2 19-230 (1989)).

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, orIgG4) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Diagnosing a Disease Associated with CDH3

The antibodies of the present invention may be used as a marker fordiagnosing a disease that is associated with CDH3 such as cancer. Morespecifically, by detecting the CDH3 polypeptide with the antibody of thepresent invention in a subject-derived sample, a disease associated withCDH3 can be diagnosed. Thus, the present invention provides methods fordiagnosing a disease associated with CDH3 or a predisposition fordeveloping the disease in a subject by detecting the CDH3 polypeptidewith the antibody of the present invention in the subject-derivedsample. The methods comprise the steps of:

(a) contacting a sample or a specimen from the subject with the antibodyor the fragment thereof of the present invention;

(b) detecting the CDH3 polypeptide in the sample or specimen; and

(c) judging whether or not the subject suffers from or is at risk ofdeveloping the disease based on the relative abundance of the CDH3protein compared to a control.

In a typical embodiment, a disease associated with CDH3 is cancer, morespecifically, pancreatic, lung, colon, prostate, breast, gastric orliver cancer.

Alternatively, in a other embodiments, the antibody of the presentinvention may be used for detecting or imaging a cancer in a livingbody. More specifically, the present invention provides methods ofdetecting or imaging a cancer which comprise the steps of:

(1) administering to a subject the antibody or fragment thereof of thepresent invention;

(2) detecting accumulation or localization of the antibody or thefragment in a living body, and

(3) determining the location of the antibody or the fragment, within thesubject.

Alternatively, according to the present invention, cancer cells ortissues may be detected in a subject. For example, the present inventionprovides methods for detecting a cancer, in which CDH3 is expressed, ina subject, comprising: administering the antibody or fragment thereof ofthe present invention to the subject allowing the antibody or fragmentspecifically binds to CDH3 polypeptide in subject cells or tissue;visualizing the antibody bound in the cells or tissue; and comparing thelevel of the antibody bound to the cells or tissue to a normal controlcells or tissue, wherein an increase in the level of the antibody boundto the subject cells or tissue relative to the normal control cells ortissue is indicative of a cancer in the subject.

Preferably, in order to trace the antibody administered into a livingbody, the antibody may be labeled with detectable molecules. Forexample, the behavior of antibodies labeled with a fluorescentsubstance, luminescent substance, or radioisotope can be traced in vivo.Methods for labeling an antibody with such molecules are well known inthe art.

Antibodies labeled with a fluorescent substance or a luminescentsubstance can be observed, for example, using an endoscope or alaparoscope. When using a radioisotope, the localization of an antibodycan be imaged by tracing the radioactivity of the radioisotope. In thepresent invention, the localization or accumulation of the antibody ofthe present invention in vivo demonstrates the presence of cancer cells.

Similar methods have been employed for other antibodies, and the skilledpractitioner will be aware of the various methods suitable for imagingthe location of detectably bound antibody or fragments within the body.As a nonlimiting guide, about 10-1000 microgram (mcg.), preferably about50-500 mcg, more preferably about 100-300 mcg, more preferably about200-300 mcg of purified antibody are administered. For example,applicable doses for humans include about 100-200 mcg/kg body weight, or350-700 mg/m² of body-surface area.

Kits for Diagnosing a Disease Associated with CDH3

The present invention provides a kit for diagnosis of a diseaseassociated with CDH3. Specifically, the kit includes the antibody orfragment thereof of the present invention as a detection regent for CDH3polypeptide. In an embodiment, the antibody for the kit of the presentinvention may be labeled with fluorescent substance, luminescentsubstance, or radioisotope. Methods for labeling antibodies anddetecting the labeled antibodies are well known in the art and anylabels and methods may be employed for the present invention.

Furthermore, the kit may include positive and negative control reagents,and a secondary antibody for detecting the antibody of the presentinvention. For example, tissue samples obtained from healthy normalsubjects or noncancerous tissues may serve as useful negative controlreagents. The kit of the present invention may further include othermaterials desirable from a commercial and user standpoint, includingbuffers, diluents, filters, needles, syringes, and package inserts(e.g., written, tape, CDROM, etc.) with instructions for use. Thesereagents and such may be retained in a container with a label. Suitablecontainers include bottles, vials, and test tubes. The containers may beformed from a variety of materials, such as glass or plastic.

In other embodiments, the present invention further provides a kit foruse in detecting, imaging or treating a cancer within a subject to bediagnosed comprising the antibody of the present invention.Alternatively, the present invention also provides a diagnostic agentcomprising the antibody of the present invention, so that the agent isused for administration into a subject to be diagnosed for a diseaseassociated with CDH3, including a cancer. In preferable embodiments, theantibody of the present invention may be labeled with a radioisotope.For example, the kit of the present invention may contain the antibodyof the present invention modified with chelating agent and radioactivesubstance. MX-DOPA is preferable chelating agent for modifying theantibody. Meanwhile, indium-111 (¹¹¹In) can be used as a tracer forbioimaging. Alternatively, in order for radioimmunotherapy of a diseaseassociated with CDH3, the antibody may be labeled with beta nuclidese.g. Yttrium-90 (⁹⁰Y). In the present invention, indium-111 (¹¹¹In) orYttrium-90 (⁹⁰Y) may also be provided as salt or solution thereof.Suitable salt of indium-111 (¹¹¹In) or Yttrium-90 (⁹⁰Y) is chloride.

In a preferable embodiment, a disease associated with CDH3 ispancreatic, lung, colon, prostate, breast, gastric or liver cancers.

Therapeutic Uses

Described below are methods and pharmaceutical compositions for treatingand/or preventing a disease associated with CDH3, or inhibitingCDH3-expressing cell growth using the antibody of the present invention.In typical embodiment, a disease associated with CDH3 is cancer,including but not limited to a pancreatic, lung, colon, prostate,breast, gastric or liver cancer cell. Specifically, the method fortreating and/or preventing a disease associated with CDH3, or inhibitingCDH3-expressing cell growth, in a subject according to the presentinvention comprises administering to the subject an effective amount ofthe antibody or fragment thereof of the present invention.

The subject in the present invention may be animals including mammalsand avian animals. For example, mammals may include humans, mice, rats,monkeys, rabbits, and dogs.

The antibody or fragment thereof described herein can specifically bindto CDH3 polypeptide, so when the antibody or fragment thereof areadministered to a subject, it binds to CDH3 polypeptide in the subjectand may suppress CDH3-expressing cell growth such as cancerous cells.Alternatively, when the antibody or fragment thereof may be conjugatedwith a therapeutic moiety and administered to a subject, it is deliveredto a region that expresses CDH3 polypeptide (i.e. suffered region) in asubject and the therapeutic moiety can be selectively delivered to thesuffered region and acted thereon. Such therapeutic moiety may be anytherapeutics that are known or will be developed for having atherapeutic efficacy on the cancer and includes, but not limited to, aradioisotope label and chemotherapeutic agent. A radioisotope labelwhich can be used as therapeutics can be selected depending on a varietyof elements including beta-ray energy and its emission efficiency, thepresence or absence of gamma-ray emitted, its energy and emissionefficiency, physical half-life, and labeling procedure. Generally, theradioisotope label based on yttrium (such as ⁹⁰Y) and iodine (such as¹²⁵I and ¹³¹I) may be used. A chemotherapeutic agent may be any agentthat is known or will be developed for treating the cancer and includes,but not limited to, methotrexate, taxol, mercaptopurine, thioguanine,cisplatin, carboplatin, mitomycin, bleomycin, doxorubicin, idarubicin,daunorubicin, dactinomycin, vinblastine, vincristine, vinorelbine,paclitaxel, and docetaxel. The antibody or fragment thereof of thepresent invention can selectively bind to CDH3 polypeptide and not bindto a normal cell, so side effect which is caused by the antibody orfragment thereof, or radioisotope or chemotherapeutic agent can beeffectively avoided and therefore the therapeutic potency may be high.

The antibody or fragment thereof described herein can be administered toa subject at effective doses to treat or prevent a CDH3-associateddisease. An effective dose refers to that amount of the antibody or afragment thereof sufficient to result in a healthful benefit in thetreated subject. Formulations and methods of administration that can beemployed when the pharmaceutical composition contains the antibody orfragment thereof of the present invention are described below.

It is to be further understood that a cocktail of different monoclonalantibodies, such as a mixture of the specific monoclonal antibodiesdescribed herein or fragments, may be administered, if necessary ordesired, to alleviate diseases associated with CDH3. Indeed, using amixture of monoclonal antibodies, or fragments thereof, in a cocktail totarget several antigens, or different epitopes, on disease cells, is anadvantageous approach, particularly to prevent evasion of tumor cellsand/or cancer cells due to downregulation of one of the antigens.

Pharmaceutical compositions for use in accordance with the presentinvention can be formulated in conventional manner using one or morepharmaceutically acceptable carriers or excipients. Thus, the presentinvention provides pharmaceutical compositions for treating orpreventing a disease associated with CDH3, or inhibiting CDH3-expressingcell growth, comprising an effective amount of the antibody or fragmentthereof of the present invention, and pharmaceutically acceptablecarriers or excipients.

The antibodies or fragments thereof can be formulated for parenteraladministration (i.e., intravenous or intramuscular) by injection, via,for example, bolus injection or continuous infusion. Formulations forinjection can be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions cantake such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the antibody can bein lyophilized powder form for constitution with a suitable vehicle,e.g., sterile pyrogen-free water, before use.

Toxicity and therapeutic efficacy of the antibody or fragment, or thetherapeutic moiety conjugated thereto can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD/ED.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosages for use in humans. The dosage ofthe antibodies lies preferably within a range of circulating plasmaconcentrations that include the ED50 with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed, the route of administration utilized and types and amounts ofthe therapeutic moiety conjugated. For the antibody or fragment thereofof the present invention, the effective dose can be estimated initiallyfrom cell culture assays. A dose can be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(i.e., the concentration of the test antibody that achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

While depending on the conditions and age of the subject and/oradministration route, one skilled in the art can select an appropriatedose of the pharmaceutical composition of the present invention. Forexample, the pharmaceutical composition of the present invention isadministered in an amount such that the antibody according to thepresent invention is administered to the subject in a day in an amountof about 3 to about 15 mcg per kg body weight of subject, and preferablyof about 10 to about 15 mcg per kg body weight of subject. Theadministration interval and times can be selected in consideration ofthe condition and age of the subject, administration route, and responseto the pharmaceutical composition. For example, the pharmaceuticalcomposition can be administered to the subject one to 5 times,preferably 1 times a day for 5 to 10 days.

In another aspect, when the composition comprising the radioisotopelabeled antibody is parenterally administered, the administrative dosefor a single adult is 0.1 mCi/kg to 1.0 mCi/kg, preferably 0.1 mCi/kg to0.5 mCi/kg, and more preferably 0.4 mCi/kg at once.

The pharmaceutical composition can be administered systemically orlocally. It is preferably administered in a targeting delivery manner soas to deliver the active component to an affected site.

In particular embodiments, the methods and compositions of the presentinvention are used for the treatment or prevention of the cancertogether with one or a combination of chemotherapeutic agents including,but not limited to, methotrexate, taxol, mercaptopurine, thioguanine,cisplatin, carboplatin, mitomycin, bleomycin, doxorubicin, idarubicin,daunorubicin, dactinomycin, vinblastine, vincristine, vinorelbine,paclitaxel, and docetaxel.

With respect to radiation therapy, any radiation therapy protocol can beused depending upon the type of the cancer to be treated. For example,but not by way of limitation, X-ray radiation can be administered. Gammaray emitting radioisotopes, such as radioactive isotopes of radium,cobalt, and other elements may also be administered to expose tissues.

In another embodiment, chemotherapy or radiation therapy isadministered, preferably at least an hour, five hours, 12 hours, a day,a week, a month, and more preferably several months (e.g., up to threemonths) subsequent to using the methods and compositions containing theantibody of the present invention. The chemotherapy or radiation therapyadministered prior to, concurrently with, or subsequent to the treatmentusing the methods and compositions according to the present inventioncan be administered by any method known in the art.

In another embodiment, the present invention also provides the use ofthe antibody of the present invention in manufacturing a pharmaceuticalcomposition for treating or preventing a disease associated with CDH3.In particular, the present invention further provides a use ofradio-labeled antibody of the present invention for manufacturing apharmaceutical composition for treating or preventing a cancer.

Alternatively, the present invention further provides the antibody ofthe present invention for use in treating or preventing a diseaseassociated with CDH3. In particular, the radio-labeled antibody of thepresent invention for use in radioimmunotherapy for cancer is alsoprovided.

Alternatively, the present invention further provides a method orprocess for manufacturing a pharmaceutical composition for treating orpreventing a disease associated with CDH3, wherein the method or processcomprises step for formulating a pharmaceutically or physiologicallyacceptable carrier with the antibody of the present invention as activeingredients. In particular, the present invention further provides amethod or process for manufacturing a pharmaceutical composition fortreating or preventing a cancer, wherein the method or process comprisesstep for formulating a pharmaceutically or physiologically acceptablecarrier with the radio-labeled antibody of the present invention asactive ingredients.

In another embodiment, the present invention also provides a method orprocess for manufacturing a pharmaceutical composition for treating orpreventing a disease associated with CDH3, wherein the method or processcomprises step for admixing an active ingredient with a pharmaceuticallyor physiologically acceptable carrier, wherein the active ingredient isthe antibody of the present invention. In particular, the presentinvention further provides a method or process for manufacturing apharmaceutical composition for treating or preventing a cancer, whereinthe method or process comprises step for admixing the radio-labeledantibody of the present invention with a pharmaceutically orphysiologically acceptable carrier.

In further embodiment, the present invention provides the antibody ofthe present invention for use in bioimaging or immunoscintigraphy forcancer within a subject to be diagnosed. Alternatively, the presentinvention provides use of the antibody of the present invention formanufacturing a diagnostic agent for bioimaging or immunoscintigraphyfor cancer within a subject. The present invention further provides amethod or process for manufacturing a diagnostic agent for bioimaging orimmunoscintigraphy for cancer within a subject, wherein the method orprocess comprises step for admixing the antibody of the presentinvention with a pharmaceutically or physiologically acceptable carrier.

All prior art references cited herein are incorporated by reference intheir entirety.

EXAMPLES

Below, the present invention is further explained based on Examples.

Materials and Methods

Antibody Production.

CDH3 gene encoded extracellular domain (SEQ ID NO: 83) was amplifiedfrom cDNA pool derived from cancer cells. The product was cloned intothe pcDNA3.1 (Invitrogen, CA). To produce CDH3-specific antibody, micewere immunized subcutaneously with the domain expression vector (17.5micro g/injection) every two weeks for a month. After the confirmationof the titer of antisera, splenocytes were extracted from the mice andfused to myeloma cells to prepare hybridomas. Hybridomas which canproduce an antibody binding to native CDH3 antigen on the surface of thecancer cells were screened. Through the screening, hybridoma clone #3,clone #4, clone #5 and clone #6 were confirmed to produceantigen-specific antibody at high level, therefore these hybridomas wereselected to produce antibody for further experiments. The hybridomaclone #3, clone #4, clone #5 and clone #6 were injectedintraperitoneally into mice, and the ascites were recovered after 2 to 3weeks. The antibodies were purified from the ascites using Protein Acolumn (GE Healthcare, NJ). Herein, antibodies are also referred to asclone #3, clone #4, clone #4 and clone #6.

Cell Culture.

H1373, human non-small cell lung cancer line, was used for thetherapeutic study in vivo since it was confirmed to express CDH3polypeptide. H1373 was purchased from American Type Culture Collection(Manassasm, Va.), and was maintained in RPMI supplemented with 10% fetalbovine serum (FBS) and 1% penicillin/streptomycin at 37 degrees C. in ahumidified atmosphere of 5% CO₂.

Radiolabeling.

Anti-CDH3 mouse monoclonal antibodies produced by hybridoma clone #3,clone #4, clone #5, clone #6 and control antibody, which was normalmouse IgG1 (Nordic immunological laboratories, Tiburg, The Netherlands),were labeled with Yttrium-90 (90Y). Antibody was labeled with 90Y via abifunctional metal ion chelating agent, pSCN-Bn-DTPA (Macrocyclics,Dallas, Tex., USA). One milligram of the antibody was conjugated to thatchelator in dimethylformamide at a molar ratio of 1:5, respectively.After incubation at 37 degrees C. for 20 hours, antibody-chelatorcomplexes were purified using Biospin Column 6 (Bio-Rad, Tokyo, Japan).90YCl₃ (QSA Global, Brauschweig, Germany) was pre-incubated with 0.25 Macetic acid (pH5.5) for 5 minutes at room temperature in parallel. Toobtain 90Y-labeled antibodies, the antibody-chelator complex wasincubated with the preincubated 90YCl₃ solution at 37 degrees C. for 1hour, respectively. Labeled antibody was purified using Biospin Column 6according to manufacture's instructions. During labeling processes,degradation of these antibodies was not observed.

Xenograft Models.

Animal care and treatment was performed in accordance with theguidelines of animal use and animal committee of the Gunma University.100 microlitter of H1373 cell suspension (1×107 cells) was inoculatedsubcutaneously into the right flank of female 3- to 5-week-old nude mice(Charles River Laboratories Japan Inc. Yokohama, Japan). These mice werekept for several weeks to develop the tumors. The established tumorswere isolated from tumor-bearing mice and dissected into cubic tissuefragments 2 mm on a side. These fragments were transplanted seriallyinto nude mice. After the transplantation, these mice were kept untilthe average tumor volume reached 100 mm³.

Radiotherapy.

Xenograft mice were randomly assigned to ten different treatment groups.90Y-labeled antibodies (4-10 mCi/mg) were prepared as described above.The mice were injected intravenously with 90Y-labeled or non-labeledclone #3, clone #4, clone #5 or clone #6. 90Y-labeled normal mouse IgG1was injected as control. Radioactivity of injected antibodies wasadjusted to 100 microCi per animal. Body weight and tumor volume of thetreated-xenograft mice were monitored for 5 weeks after injection. Thetumor volume (mm³) was calculated using following formula: (the shortestdiameter)²×(the longest diameter)×0.5.

Reduced SDS-PAGE.

Each 5 micro-g of anti-CDH3 antibody was mixed with SDS buffer thatincluded 4% SDS, 125 mM Tris-HCl (pH6.8), 20% glycerol, 0.04%Bromophenol Blue and 10% mercaptoethanol. After heated, the mixtureswere applied to 4-20% gradient SDS-PAGE gel. Then, the gel was stainedwith Coomassie brilliant blue R-250 (CBB) and destained by using 10%methanol and 7% acetic acid. An image of the gel was captured byscanner.

Analysis for Amino Acid Sequence of Variable Region.

Total RNAs were extracted from hybridoma clone #3, clone #4 and clone #5using RNeasy Mini Kit (QIAGEN). The cDNAs were synthesized from thetotal RNAs using SuperScript II Reverse Transcriptase (Invitrogen). Thepolynucleotides encoding variable regions of monoclonal antibodies wereamplified using NovaTaq DNA polymerase (Novagen) and Mouse Ig-Primer Set(Novagen). The primers for amplification are follows:

MuIgVH5′-B; (SEQ ID NO: 84) 5′-GGGAATTCATGRAATGSASCTGGGTYWTYCTCTT-3′for heavy chain 5′ primer, MuIg kappa VL5′-D (mixture of following primers); (SEQ ID NO: 85)5′-ACTAGTCGACATGAGGRCCCCTGCTCAGWTTYTTGGIWTCTT-3′ and (SEQ ID NO: 86)5′-ACTAGTCGACATGGGCWTCAAGATGRAGTCACAKWYYCWGG-3′ for light chain 5′primer, MuIgGVH3′-2; (SEQ ID NO: 87)5′-CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG-3′ for heavy chain 5′ primer, andMuIg kappa VL3′-1; (SEQ ID NO: 88) 5′-CCCAAGCTTACTGGATGGTGGGAAGATGGA-3′for light chain 3′ primer.

PCR products were cloned into pCR2.1-TOPO (Invitrogen). Insert regionswere sequenced and the nucleic acid sequences of the variable regions(except for the signal sequence) of clone #3, clone #4 and clone #5 weredetermined.

Following symbols are used for the different nucleotides in the primersequences; B as C, G or T, D as A, G or T, H as A, C or T, I as inosine,K as G or T, M as A or C, R as A or G, S as C or G, V as A, C or G, W asA or T and Y as C or T.

Results.

To evaluate the efficacy of CDH3 targeted radioimmunotherapy, anti-CDH3antibodies were radiolabeled with beta-emmitting isotope ⁹⁰Y (t1/2=64.1hours), and administered by intravenous injection to tumor-bearing nudemice. The growth rate of tumors treated with yttrium-90 labeled clone#3, #4 and #6 were drastically decreased by radiation from yttrium-90conjugated with the antibodies (FIG. 1). In particular, 90Y-labeledclone #3 and clone #6 strongly suppressed tumor growth in H1373xenograft mice during observation. On the other hand, ⁹⁰Y-labeledcontrol antibody showed no effect on tumor growth. Therefore,therapeutic effects of ⁹⁰Y-labeled antibodies seemed to depend on itsaffinities to CDH3 polypeptide, which was expressed on tumor cellsurface. The body weights of mice treated with any antibodies did notsignificantly decrease (data not shown).

Anti-CDH3 antibody clone #3, clone #4 and clone #6 conjugated yttrium-90exerted remarkable therapeutic effects against tumor. Therefore, CDH3would be an attractive target for cancer therapy and anti-CDH3 antibodywould be available as a novel tool for cancer therapy.

The amino acid sequences of H chain V regions and L chain Variableregions of mouse monoclonal antibodies were determined as follows:

clone #3, H chain variable region (except for the signal sequence):(SEQ ID NO: 4) QVQLQQPGAELVRPGSSVKLSCKASGYTFTSFWIHWVKQRPMQGLEWIGNIDPSDSETHYNQYFKDRATLTVDRSSSTAYMHLTSLTSEDSAVYYCARGG TGFSSWGQGTLVTVSA(encoded by the nucleic acid sequence shown in  SEQ ID NO: 3);clone #3, L chain variable region (except for the signal sequence):(SEQ ID NO: 12) DIKMTQSPSSMYASLGERVTITCKASQDIDSYLSWFQQKPGKSPKTLIHRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPRTFGG GTKLEIK(encoded by the nucleic acid sequence shown in SEQ ID NO: 11);clone #4, H chain variable region (except for the signal sequence):(SEQ ID NO: 20) LVQLQQPGAELVRPGSSVKLSCKTSGYTFTSYWMHWIKQRPIQGLEWIGNIDPSDSETHYNQNFNDRATFTVDKSSSTAYMELSSLTSEDSAVYYCARGG TGFAYWGQGTLVTVSA(encoded by the nucleic acid sequence shown in SEQ ID NO: 19);clone #4, L chain variable region (except for the signal sequence):(SEQ ID NO: 28) DIKMTQSPSSMYASLGERVTITCKASQDINNYLGWFQQKPGKSPKTLIHRTDRLIEGVPSRFSGSGSGQDYSLTISSLEYEDVGTYYCLQYDEFPRMFGG GTKLEIK(encoded by the nucleic acid sequence shown in SEQ ID NO: 27);Clone #5, H chain variable region (except for the signal sequence):(SEQ ID NO: 36) LVQLQQPGAELVRPGSSVKLSCKASGYTFTSYWMHWIKQRPIQGLEWIGNIDPSDSETHYNQKFNDRARLTVDKSSSTAYMHLSSLTSEDSAVYYCARGG TGFAYWGQGTLVTVSA(encoded by the nucleic acid sequence shown in SEQ ID NO: 35); andclone #5, L chain variable region (except for the signal sequence):(SEQ ID NO: 44) DIKMTQSPSSMYASLGERVTITCKASQDINNYLGWFQQKPGKSPKTLIHRTDRLIEGVPSRFSGSGSGQDYSLTISSLEYEDVGTYYCLQYDEFPRMFGG GTKLDIK(encoded by the nucleic acid sequence shown in SEQ ID NO: 43).

The CDR sequences of the antibodies determined by the Kabat definitionare as follows: clone #3, SFWIH (SEQ ID NO: 6) (encoded by the nucleicacid sequence shown in SEQ ID NO: 5) as VH CDR1, NIDPSDSETHYNQYFKD (SEQID NO: 8) (encoded by the nucleic acid sequence shown in SEQ ID NO: 7)as VH CDR2 and GGTGFSS (SEQ ID NO: 10) (encoded by the nucleic acidsequence shown in SEQ ID NO: 9) as VH CDR3, KASQDIDSYLS (SEQ ID NO: 14)(encoded by the nucleic acid sequence shown in SEQ ID NO: 13) as VLCDR1, RANRLVD (SEQ ID NO: 16) (encoded by the nucleic acid sequenceshown in SEQ ID NO: 15) as VL CDR2 and LQYDEFPRT (SEQ ID NO: 18)(encoded by the nucleic acid sequence shown in SEQ ID NO: 17) as VLCDR3;

clone #4, SYWMH (SEQ ID NO: 22) (encoded by the nucleic acid sequenceshown in SEQ ID NO: 21) as VH CDR1, NIDPSDSETHYNQNFND (SEQ ID NO: 24)(encoded by the nucleic acid sequence shown in SEQ ID NO: 23) as VH CDR2and GGTGFAY (SEQ ID NO: 26) (encoded by the nucleic acid sequence shownin SEQ ID NO: 25) as VH CDR3, KASQDINNYLG (SEQ ID NO: 30) (encoded bythe nucleic acid sequence shown in SEQ ID NO: 29) as VL CDR1, RTDRLIE(SEQ ID NO: 32) (encoded by the nucleic acid sequence shown in SEQ IDNO: 31) as VL CDR2 and LQYDEFPRM (SEQ ID NO: 34) (encoded by the nucleicacid sequence shown in SEQ ID NO: 33) as VL CDR3; and

clone #5, SYWMH (SEQ ID NO: 38) (encoded by the nucleic acid sequenceshown in SEQ ID NO: 37) as VH CDR1, NIDPSDSETHYNQKFNDRA (SEQ ID NO: 40)(encoded by the nucleic acid sequence shown in SEQ ID NO: 39) as VH CDR2and GGTGFAY (SEQ ID NO: 42) (encoded by the nucleic acid sequence shownin SEQ ID NO: 41) as VH CDR3, KASQDINNYLG (SEQ ID NO: 46) (encoded bythe nucleic acid sequence shown in SEQ ID NO: 45) as VL CDR1, RTDRLIE(SEQ ID NO: 48) (encoded by the nucleic acid sequence shown in SEQ IDNO: 47) as VL CDR2 and LQYDEFPRM (SEQ ID NO: 50) (encoded by the nucleicacid sequence shown in SEQ ID NO: 49) as VL CDR3.

SDS-PAGE analysis was performed under reducing condition. The bandpatterns on the gel were characterized by molecular weight range 40-50kDa which corresponding to IgG heavy chain and lower molecular weightranges of 20-30 kDa which correspond to IgG light chain. Anti-CDR3antibody clone #3, clone #4 and clone #5 exhibited single heavy chainband and single light chain band as general IgG. On the other hand,anti-CDR3 antibody clone #6 showed two heavy chain bands and singlelight chain band. Incomplete glycosylation at variable region of heavychain caused to the additional heavy chain band during reduced SDS-PAGE.As shown in FIG. 2, incomplete glycosylation affects uniformity of theantibody, and it may create difficulty in development of therapeuticdrugs. Therefore, variants of clone #6 which have a single amino acidsubstitution at glycosylation site of clone #6 were designed in order toavoid glycosylation in H chain variable region. These variants would bemore applicable for development of antibody-based cancer drug than clone#6.

The amino acid sequences of H chain variable regions of clone #6variants are as follows (underline indicates a substituted amino acidresidue):

clone #6NS, H-chain variable region (except for the signal sequence):(SEQ ID NO: 68) QVQLQQPGAELVKPGTSVKLSCKSSGYTFTSYWIHWVKQRPGHGLEWIGEIDPSDSYTYYNQNFKGKATLTIDKSSSTAYMQLNSLTSEDSAVFYCARSG YGNLFVYWGQGTLVTVSA(encoded by the nucleic acid sequence shown in SEQ ID NO: 67);clone #6NT, H-chain variable region (except for the signal sequence):(SEQ ID NO: 72) QVQLQQPGAELVKPGTSVKLSCKSSGYTFTSYWIHWVKQRPGHGLEWIGEIDPSDTYTYYNQNFKGKATLTIDKSSSTAYMQLNSLTSEDSAVFYCARSG YGNLFVYWGQGTLVTVSA(encoded by the nucleic acid sequence shown in SEQ ID NO: 71);clone #6NA, H-chain variable region (except for the signal sequence):(SEQ ID NO: 76) QVQLQQPGAELVKPGTSVKLSCKSSGYTFTSYWIHWVKQRPGHGLEWIGEIDPSDAYTYYNQNFKGKATLTIDKSSSTAYMQLNSLTSEDSAVFYCARSG YGNLFVYWGQGTLVTVSA(encoded by the nucleic acid sequence shown in SEQ ID NO: 75); andclone #6NQ, H-chain variable region (except for the signal sequence):(SEQ ID NO: 80) QVQLQQPGAELVKPGTSVKLSCKSSGYTFTSYWIHWVKQRPGHGLEWIGEIDPSDQYTYYNQNFKGKATLTIDKSSSTAYMQLNSLTSEDSAVFYCARSG YGNLFVYWGQGTLVTVSA(encoded by the nucleic acid sequence shown in SEQ ID NO: 79).

The amino acid sequences of L chain variable regions (except for thesignal sequence) of clone #6 variants are the same as that of clone #6;

(SEQ ID NO: 60) QIVLTQSPAIMSSSPGEKVTMSCSATSSVTYMYWYQQKPGSSPKPWIFRTSNLASGVPTRFSGSGSGTSYSLTISSMEAEDAATYYCQHYHIYPRTFGGG TKLEIK(encoded by the nucleic acid sequence shown in SEQ ID NO: 59).

The VH CDR2 sequences determined by the Kabat definition of the clone #6variants are; EIDPSDSYTYYNQNFKG (SEQ ID NO: 70) (encoded by the nucleicacid sequence shown in SEQ ID NO: 69) for clone #6NS, EIDPSDTYTYYNQNFKG(SEQ ID NO: 74) (encoded by the nucleic acid sequence shown in SEQ IDNO: 73) for clone #6NT, EIDPSDAYTYYNQNFKG (SEQ ID NO: 78) (encoded bythe nucleic acid sequence shown in SEQ ID NO: 77) for clone #6NA, andEIDPSDQYTYYNQNFKG (SEQ ID NO: 82) (encoded by the nucleic acid sequenceshown in SEQ ID NO: 81) for clone #6NQ.

The other CDR sequences determined by the Kabat definition of thevariants are the same as those of clone #6; SYWIH (SEQ ID NO: 54)(encoded by the nucleic acid sequence shown in SEQ ID NO: 53) as VHCDR1, SGYGNLFVY (SEQ ID NO: 58) (encoded by the nucleic acid sequenceshown in SEQ ID NO: 57) as VH CDR3, SATSSVTYMY (SEQ ID NO: 62) (encodedby the nucleic acid sequence shown in SEQ ID NO: 61) as VL CDR1, RTSNLAS(SEQ ID NO: 64) (encoded by the nucleic acid sequence shown in SEQ IDNO: 63) as VL CDR2 and QHYHIYPRT (SEQ ID NO: 66) (encoded by the nucleicacid sequence shown in SEQ ID NO: 65) as VL CDR3.

INDUSTRIAL APPLICABILITY

The present invention is based, at least in part, on the discovery thata cancer expressing CDH3 can be treated with radioisotope labeledanti-CDH3 antibody in vivo. CDH3 was reported as a gene stronglyexpressed in pancreatic, lung, colon, prostate, breast, gastric or livercancers. Thus, treatment of a cancer, for example, pancreatic, lung,colon, prostate, breast, gastric or liver cancer is conveniently carriedout using anti-CDH3 antibodies labeled with radioisotope label.

1. An antibody or a fragment thereof, wherein the antibody comprises anH (heavy) chain V (variable) region and an L (light) chain V region,wherein the H chain V region and the L chain V region are selected fromthe group consisting of: (a) an H chain V region comprisingcomplementarity determining regions (CDRs) included in an H chain Vregion having the amino acid sequence shown in SEQ ID NO: 4 or CDRsfunctionally equivalent thereto, and an L chain V region comprising CDRsincluded in an L chain V region having the amino acid sequence shown inSEQ ID NO 12 or CDRs functionally equivalent thereto; (b) an H chain Vregion comprising CDRs included in an H chain V region having the aminoacid sequence shown in SEQ ID NO: 20 or CDRs functionally equivalentthereto, and an L chain V region comprising CDRs included in an L chainV region having the amino acid sequence shown in SEQ ID NO: 28 or CDRsfunctionally equivalent thereto; (c) an H chain V region comprising CDRsincluded in an H chain V region having the amino acid sequence shown inSEQ ID NO: 36 or CDRs functionally equivalent thereto, and an L chain Vregion comprising CDRs included in an L chain V region having the aminoacid sequence shown in SEQ ID NO: 44 or CDRs functionally equivalentthereto; and (d) an H chain V region comprising CDRs included in an Hchain V region having the amino acid sequence shown in SEQ ID NOs: 68,72, 76 or 80, or CDRs functionally equivalent thereto, and an L chain Vregion comprising CDRs included in an L chain V region having the aminoacid sequence shown in SEQ ID NO: 60 or CDRs functionally equivalentthereto, and wherein the antibody is capable of binding to a CDR3polypeptide or a partial peptide thereof.
 2. The antibody or a fragmentthereof according to claim 1, wherein the H chain V region and the Lchain V region are selected from the group consisting of: (a) an H chainV region comprising CDR1 having the amino acid sequence shown in SEQ IDNO: 6, CDR2 having the amino acid sequence shown in SEQ ID NO: 8 andCDR3 having the amino acid sequence shown in SEQ ID NO: 10, and an Lchain V region comprising CDR1 having the amino acid sequence shown inSEQ ID NO: 14, CDR2 having the amino acid sequence shown in SEQ ID NO:16 and CDR3 having the amino acid sequence shown in SEQ ID NO: 18; (b)an H chain V region comprising CDR1 having the amino acid sequence shownin SEQ ID NO: 22, CDR2 having the amino acid sequence shown in SEQ IDNO: 24 and CDR3 having the amino acid sequence shown in SEQ ID NO: 26,and an L chain V region comprising CDR1 having the amino acid sequenceshown in SEQ ID NO: 30, CDR2 having the amino acid sequence shown in SEQID NO: 32 and CDR3 having the amino acid sequence shown in SEQ ID NO:34; (c) an H chain V region comprising CDR1 having the amino acidsequence shown in SEQ ID NO: 38, CDR2 having the amino acid sequenceshown in SEQ ID NO: 40 and CDR3 having the amino acid sequence shown inSEQ ID NO: 42, and an L chain V region comprising CDR1 having the aminoacid sequence shown in SEQ ID NO: 46, CDR2 having the amino acidsequence shown in SEQ ID NO: 48 and CDR3 having the amino acid sequenceshown in SEQ ID NO: 50; and (d) an H chain V region comprising CDR1having the amino acid sequence shown in SEQ ID NO: 54, CDR2 having theamino acid sequence shown in SEQ ID NOs: 70, 74, 78 or 82 and CDR3having the amino acid sequence shown in SEQ ID NO: 58, and an L chain Vregion comprising CDR1 having the amino acid sequence shown in SEQ IDNO: 62, CDR2 having the amino acid sequence shown in SEQ ID NO: 64 andCDR3 having the amino acid sequence shown in SEQ ID NO:
 66. 3. Theantibody or fragment thereof according to claim 1, wherein the antibodyis selected from the group consisting of a mouse antibody, a chimericantibody, a humanized antibody, an antibody fragment, and single-chainantibody.
 4. The antibody or fragment thereof according to claim 3,wherein the antibody comprises an H chain V region having the amino acidsequence selected from the group consisting of SEQ ID NOs: 4, 20, 36,68, 72, 76 and 80 and/or an L chain V region having the amino acidsequence selected from the group consisting of SEQ ID NOs: 12, 28, 44and
 60. 5. The antibody or fragment thereof according to claim 4,wherein the antibody comprises: (a) an H chain V region having the aminoacid sequence shown in SEQ ID NO: 4 and an L chain V region having theamino acid sequence shown in SEQ ID NO: 12; (b) an H chain V regionhaving the amino acid sequence shown in SEQ ID NO: 20 and an L chain Vregion having the amino acid sequence shown in SEQ ID NO: 28; (c) an Hchain V region having the amino acid sequence shown in SEQ ID NO: 36 andan L chain V region having the amino acid sequence shown in SEQ ID NO:44; or (d) an H chain V region having the amino acid sequence shown inSEQ ID NOs: 68, 72, 76 or 80 and an L chain V region having the aminoacid sequence shown in SEQ ID NO:
 60. 6. The antibody or fragmentthereof according to claim 4, wherein the antibody is chimeric antibody.7. The antibody or fragment thereof according to claim 6, wherein theantibody is a humanized antibody.
 8. The antibody or fragment thereofaccording to claim 7, wherein the humanized antibody further comprises ahuman antibody FR (framework) region and/or a human antibody C region.9. The antibody or fragment thereof according to claim 1, wherein theantibody is conjugated with a cytotoxic, a therapeutic agent, aradioisotope label or a fluorescent label.
 10. The antibody or fragmentthereof according to claim 9, wherein the radioisotope label is selectedfrom 90yttrium (⁹⁰Y) and 111 indium (¹¹¹In).
 11. A method for treatingor preventing a disease associated with CDH3, or inhibitingCDH3-expressing cell growth, in a subject, wherein the method comprisesadministering to the subject an effective amount of the antibody orfragment according to claim
 1. 12. A method for diagnosis of a diseaseassociated with CDH3 or of a predisposition to develop the disease in asubject, wherein the method comprises: (a) contacting a sample or aspecimen from the subject with the antibody or fragment according toclaim 1; (b) detecting a CDH3 polypeptide in the sample or specimen; and(c) judging whether or not the subject suffers from or is at risk ofdeveloping the disease based on the relative abundance of the CDH3polypeptide compared to a control.
 13. A pharmaceutical composition fortreating or preventing a disease associated with CDH3, or inhibitingCDH3-expressing cell growth, wherein the pharmaceutical compositioncomprises an effective amount of the antibody or fragment according toclaim 1 and a pharmaceutically acceptable carrier or excipient.
 14. Akit for diagnosis of a disease associated with CDH3, wherein the kitcomprises the antibody or fragment according to claim 1.